Probes for improved melt discrimination and multiplexing in nucleic acid assays

ABSTRACT

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.

This application is a continuation of U.S. patent application Ser. No.15/656,541, filed Jul. 21, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/823,288, filed Aug. 11, 2015, now U.S. Pat. No.9,982,291, which claims the benefit of U.S. Provisional PatentApplication No. 62/035,783, filed Aug. 11, 2014, the entirety of whichis incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“LUMNP0129ST25.txt”, which is 8 KB (as measured in Microsoft Windows®)and was created on Aug. 1, 2020, is filed herewith by electronicsubmission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns the detection of nucleic acids.

2. Description of Related Art

Polymerase chain reaction (PCR) is a molecular biology technique forenzymatically replicating DNA without using a living organism. PCR iscommonly used in medical and biological research labs for a variety oftasks, such as the detection of hereditary diseases, the identificationof genetic fingerprints, the diagnosis of infectious diseases, thecloning of genes, paternity testing, and DNA computing. PCR has beenaccepted by molecular biologists as the method of choice for nucleicacid detection because of its unparalleled amplification and precisioncapability. DNA detection is typically performed at the end-point, orplateau phase of the PCR reaction, making it difficult to quantify thestarting template. Real-time PCR or kinetic PCR advances the capabilityof end-point PCR analysis by recording the amplicon concentration as thereaction progresses. Amplicon concentration is most often recorded via afluorescent signal change associated with the amplified target.Real-time PCR is also advantageous over end-point detection in thatcontamination is limited because it can be performed in a closed system.Other advantages include greater sensitivity, dynamic range, speed, andfewer processes required.

Several assay chemistries have been used in real-time PCR detectionmethods. These assay chemistries include using double-stranded DNAbinding dyes, dual-labeled oligonucleotides, such as hairpin primers,and hairpin probes. However, a drawback of current real-time PCR is itslimited multiplexing capability. Current real-time PCR technologies usereporter fluorochromes that are free in solution. This designnecessitates the use of spectrally distinct fluorochromes for each assaywithin a multiplex reaction. For example, a multiplex reaction designedto detect 4 target sequences would require an instrument capable ofdistinguishing 4 different free floating fluorochromes by spectraldifferentiation, not including controls. These requirements not onlylimit the practical multiplexing capability, but also increase costssince such instruments typically require multiple emission sources,detectors, and filters. Current real-time PCR technologies havemultiplexing capabilities from about 1-6 plex.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods for amplificationand detection of DNA. In particular, embodiments of the presentinvention provide systems and methods that greatly increase multiplexingcapabilities of detectable probes for use in detecting amplified targetsequences.

In a first embodiment, a method is provided for detecting the presenceof a target nucleic acid comprising: (a) contacting a sample with acleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising a label; (ii) a second sequence region; (iii)a sequence that is the reverse complement of the second sequence region;and (iv) a sequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of the target nucleicacid; (b) contacting the cleavable probe with an endoribonuclease,thereby cleaving probe that is hybridized with target nucleic acid toform a truncated cleavable probe; (c) allowing the truncated cleavableprobe to hybridize to itself to form a hairpin probe; (d) extending thehairpin probe; and (e) detecting the target nucleic acid by detecting achange in signal from the label. In certain aspects, the label in firstsequence region (i) is: a reporter-quencher pair and extension of thehairpin probe on the first sequence region changes the distance betweenthe reporter and quencher; or at least one non-natural nucleotidelabeled with a first member of a reporter-quencher pair and extension ofthe hairpin probe on the first sequence region results in theincorporation of a complementary non-natural nucleotide labeled with asecond member of the reporter-quencher pair. In certain aspects, all of,a portion of, or none of the sequence that is the reverse complement ofthe second sequence region (iii) may be complimentary to a first regionon a first strand of the target nucleic acid. In some embodiments, themethod further comprises performing a melt analysis on the hairpinprobe.

In one embodiment, a method is provided for detecting the presence of atarget nucleic acid comprising: (a) contacting the sample with acleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising at least one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of the target nucleic acid; (b) contacting the cleavable probewith an endoribonuclease, thereby cleaving probe that is hybridized withtarget nucleic acid to form a truncated cleavable probe; (c) allowingthe truncated cleavable probe to hybridize to itself to form a hairpinprobe; (d) extending the hairpin probe in the presence of a non-naturalnucleotide labeled with a second member of the reporter-quencher pairthat is capable of base-pairing with the at least one non-naturalnucleotide of the first sequence region; and (e) detecting the targetnucleic acid by detecting a change in signal from the label on thecleavable probe and the hairpin probe. In certain aspects, a portion ofthe sequence that is the reverse complement of the second sequenceregion (iii) may be complimentary to a first region on a first strand ofthe target nucleic acid. In some embodiments, the method furthercomprises performing a melt analysis on the hairpin probe.

In another embodiment, a method is provided for detecting the presenceof a target nucleic acid comprising: (a) contacting a sample with acleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region labeled with a reporter-quencher pair; (ii) a secondsequence region; (iii) a sequence that is the reverse complement of thesecond sequence region; and (iv) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of the target nucleic acid; (b) contacting the cleavable probewith an endoribonuclease, thereby cleaving the cleavable probe that ishybridized with the target nucleic acid to form a truncated probe; (c)allowing the truncated probe to hybridize to itself to form a hairpinprobe; (d) extending the hairpin probe onto the first sequence regionsuch that the distance between the reporter and quencher is increased;and (e) detecting the target nucleic acid by detecting a change insignal from the reporter. In certain embodiments, one member of thereporter-quencher pair is at the 5′ end of the cleavable probe. In someembodiments, one member of the reporter-quencher pair is at the 5′ endof the first sequence region and the other member of thereporter-quencher pair is at the 3′ end of the first sequence region. Incertain embodiments, the reporter is a fluorescent dye. In certainaspects, all of, a portion of, or none of the sequence that is thereverse complement of the second sequence region (iii) may becomplimentary to a first region on a first strand of the target nucleicacid. In some embodiments, the method further comprises performing amelt analysis on the hairpin probe.

In certain aspects, the cleavable probe may further comprise (v) a loopsequence of one or more nucleotides between the second sequence regionand the sequence that is the reverse complement of the second sequenceregion. In some aspects, the loop sequence may be 4-20, 6-15 or 10-15nucleotides in length. In some aspects, the loop sequence may compriseat least 3-5 consecutive A nucleotides. In some embodiments, the loopsequence comprises one or more polymerase extension blocking moieties.In certain aspects the loop sequence may comprise a combination of oneor more nucleotides and one or more extension blocking moieties.Polymerase extension blocking moieties may be used as part or all of aloop sequence. Examples of extension blocking moieties include carbonspacers. Carbon spacers may include spacers that may be 3 to 36 carbonatoms in length. Common examples of internal oligonucleotide carbonspacers include spacers that are 3, 9, and 18 carbon atoms in length.Carbon spacers may be used to prevent the cleavable probes from formingnon-specific double stranded PCR products. Carbon spacers may also beused to adjust the melt temperature (Tm) of the hairpin probe. Otherpolymerase extension blocking moieties may include non-naturalnucleotides, ribonucleotides, or any other non-nucleotide chemicalmoiety.

In certain aspects, the second sequence region may be 6-20 nucleotidesin length. In certain aspects, the second sequence region compliment maybe 6-20 nucleotides in length. In certain aspects, the first sequenceregion may be 4-20 nucleotides in length. In certain aspects, thesequence that is complimentary to a first region on a first strand ofthe target nucleic acid may be 6-50, 10-50, or 6-30 nucleotides inlength. In certain aspects, the one or more ribonucleotide bases of thecleavable probe may be positioned just 3′ of the sequence that is thereverse complement of the second sequence region (also referred toherein as the second sequence region compliment). In certain aspects,the one or more ribonucleotide bases of the cleavable probe may bepositioned at least 4 bases from the 3′ end of the sequence that iscomplimentary to a first region on a first strand of the target nucleicacid. As mentioned above, all of, a portion of, or none of the sequencethat is the reverse complement of the second sequence region may becomplimentary to a first region on a first strand of the target nucleicacid.

In certain aspects, the cleavable probe may comprise a sequencecomprising 1 to 5 ribonucleotide bases that is complimentary to a firstregion on a first strand of the target nucleic acid sequence. In someaspects, the cleavable probe may comprise a sequence comprising 3 to 5ribonucleotide bases that is complimentary to a first region on a firststrand of the target nucleic acid sequence.

In certain aspects, the cleavable probe may comprise non-base pairingmodifications, which may be placed 3′ and/or 5′ of the ribobase andwithin the sequence of that probe that is otherwise complimentary to thefirst region on the first strand of the target nucleic acid. Thesemodifications may include natural or non-natural nucleotides that do notbase pair with the target sequence, or may include carbon spacers orother non-nucleotide chemical moieties. Placing non-base pairingmodifications upstream or downstream of the ribonucleotide, but within aregion of the probe that is otherwise complimentary to the targetsequence, may improve specificity of the cleavable probe. The non-basepairing moiety may be positioned between 2 and 20 nucleotides upstreamor downstream from the ribonucleotide. In certain embodiments, thenon-base pairing moiety is placed 1, 2, 3, 4, or 5, or any rangetherein, nucleotides upstream or downstream of the ribonucleotide.

In certain aspects, the at least a one non-natural nucleotide labeledwith a first member of a reporter-quencher pair may be positioned at the5′ end of the cleavable probe. In certain aspects, the cleavable probemay comprise an extension-blocking modification at the 3′ end. Incertain aspects, the second sequence region of the cleavable probe maycomprise a G/C content of at least 50%. In certain aspects, the secondsequence region of the cleavable probe may be 6-15 nucleotides inlength.

In certain aspects, the second sequence region of the cleavable probemay comprise one or more non-natural bases. In certain aspects, afterendoribonuclease or 5′-nuclease cleavage, the truncated cleavable probeand the target nucleic acid may have a melt point of less than 55° C.

In certain aspects, the first member of a reporter-quencher pair may bea reporter. In certain aspects, the reporter for use in the instantembodiments may be a fluorophore. Accordingly, in some cases, a changeis the signal may be a decrease in a fluorescent signal. In certainaspects, detecting a change in signal from the label may comprisedetecting a change (or rate of change) in signal from a reporter, suchas unquenching of a signal, as the temperature of the sample is changed.In some aspects, detecting a change in signal from the reporter maycomprise detecting a change in signal from the reporter as thetemperature of the sample is increased above (or decreased below) themelt point of hairpin probe.

In certain aspects, the cleavable probe may be attached to a solidsupport.

Certain aspects of the embodiments concern the use of at least onenon-natural nucleotide (iv). In some aspects, the non-natural nucleotideis an isobase, such as iso-guanine (isoG) or iso-cytosine (isoC). Incertain aspects, the at least one non-natural nucleotide or thequencher-labeled non-natural nucleotide may be isoG and the other may beisoC.

In a further aspect, the method may comprise (a) contacting the samplewith a second (or further) cleavable probe, said probe comprising, from5′ to 3′, (i) a first sequence region comprising at least a onenon-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a second sequence region; (iii) a sequencethat is the reverse complement of the second sequence region; and (iv) asequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of a second (orfurther) target nucleic acid; (b) contacting the cleavable probe with anendoribonuclease, thereby cleaving probe that is hybridized with targetnucleic acid to form a truncated cleavable probe; (c) allowing thetruncated cleavable probe to hybridize to itself to form a hairpinprobe; (d) extending the hairpin probe in the presence of a non-naturalnucleotide labeled with a second member of a reporter-quencher pair thatis capable of base-pairing with the at least one non-natural nucleotideof the first sequence region; and (e) detecting the second (or further)target nucleic acid by detecting a change in signal from the label onthe cleavable probe and the hairpin probe. For example, detecting thepresence of the first and/or second target nucleic acid may be performedsequentially or essentially simultaneously. In still a further aspect,the first and second probes may comprise distinguishable reporters. Inanother aspect, the first and second probes may comprise the samereporter and, in some cases, the first and second probes comprisehairpins with distinguishable melt points (e.g., melt points that differby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C. from one another, orany range derivable therein).

In still a further aspect, the method is a multiplex method andcomprises (a) contacting the sample with a third, fourth, fifth or sixthcleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising at least a one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of a third, fourth, fifth or sixth target nucleic acid; (b)contacting the cleavable probe with an endoribonuclease, therebycleaving probe that is hybridized with target nucleic acid to form atruncated cleavable probe; (c) allowing the truncated cleavable probe tohybridize to itself to form a hairpin probe; (d) extending the hairpinprobe in the presence of a non-natural nucleotide labeled with a secondmember of a reporter-quencher pair that is capable of base-pairing withthe at least one non-natural nucleotide of the first sequence region;and (e) detecting the third, fourth, fifth or sixth target nucleic acidby detecting a change in signal from the label on the cleavable probeand the hairpin probe. For example, detecting the presence of the firstand/or second target nucleic acid may be performed sequentially oressentially simultaneously. In still a further aspect, the first andsecond probes may comprise distinguishable reporters. In another aspect,the first and second probes may comprise the same reporter and, in somecases, the hairpin probes formed by the first and second probes maycomprise distinguishable melt points (e.g., melt points that differ by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C. from one another, or anyrange derivable therein). In one aspect, the hairpin probes formed bythe first, second, third, fourth, fifth, and/or sixth probes each maycomprise a distinguishable label or a distinguishable melt point.

In another embodiment, the method may comprise (a) contacting the samplewith a second (or further) cleavable probe, said probe comprising, from5′ to 3′, (i) a first sequence region comprising a reporter-quencherpair; (ii) a second sequence region; (iii) a sequence that is thereverse complement of the second sequence region; and (iv) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of a second (or further) target nucleicacid; (b) contacting the cleavable probe with an endoribonuclease,thereby cleaving probe that is hybridized with target nucleic acid toform a truncated cleavable probe; (c) allowing the truncated cleavableprobe to hybridize to itself to form a hairpin probe; (d) extending thehairpin probe onto the first sequence region; and (e) detecting thesecond (or further) target nucleic acid by detecting a change in signalfrom the reporter. For example, detecting the presence of the firstand/or second target nucleic acid may be performed sequentially oressentially simultaneously. In still a further aspect, the first andsecond probes may comprise distinguishable reporters. In another aspect,the first and second probes may comprise the same reporter and, in somecases, the first and second probes comprise hairpins withdistinguishable melt points (e.g., melt points that differ by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 ° C. from one another, or any rangederivable therein).

In still a further aspect, the method is a multiplex method andcomprises (a) contacting the sample with a third, fourth, fifth or sixthcleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising at least a one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of a third, fourth, fifth or sixth target nucleic acid; (b)contacting the cleavable probe with an endoribonuclease, therebycleaving probe that is hybridized with target nucleic acid to form atruncated cleavable probe; (c) allowing the truncated cleavable probe tohybridize to itself to form a hairpin probe; (d) extending the hairpinprobe in the presence of a non-natural nucleotide labeled with a secondmember of a reporter-quencher pair that is capable of base-pairing withthe at least one non-natural nucleotide of the first sequence region;and (e) detecting the third, fourth, fifth or sixth target nucleic acidby detecting a change in signal from the label on the cleavable probeand the hairpin probe. For example, detecting the presence of the firstand/or second target nucleic acid may be performed sequentially oressentially simultaneously. In still a further aspect, the first andsecond probes may comprise distinguishable reporters. In another aspect,the first and second probes may comprise the same reporter and, in somecases, the hairpin probes formed by the first and second probes maycomprise distinguishable melt points (e.g., melt points that differ by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C. from one another, or anyrange derivable therein). In one aspect, the hairpin probes formed bythe first, second, third, fourth, fifth, and/or sixth probes each maycomprise a distinguishable label or a distinguishable melt point.

Thus, in some further aspects, a multiplex method according to theembodiments can comprise the use of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 or more distinct probes, or any rangederivable therein, wherein each probe comprises either (1) adistinguishable melt point or (2) a distinguishable label, such that thesignal from each distinct probe may be individually discerned. In oneaspect, the first, second, third, fourth, fifth and/or sixth cleavableprobes each may comprise the same first sequence region, second sequenceregion and/or the same loop sequence between the second sequence regionand the sequence that is the reverse complement of the second sequenceregion. In certain embodiments, the loop region may comprise one or morepolymerase extension blocking moieties.

In a further embodiment, a composition is provided comprising at least afirst cleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising a label; (ii) a second sequence region; (iii)a sequence that is the reverse complement of the second sequence region;and (iv) a sequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of the target nucleicacid. In certain aspects, the comprising may further comprise areporter-labeled or quencher-labeled non-natural nucleotide. In certainembodiments, the label in first sequence region (i) is areporter-quencher pair or at least one non-natural nucleotide labeledwith a first member of a reporter-quencher pair.

In one embodiment, a composition is provided comprising at least a firstcleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising a at least one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of the target nucleic acid. In certain aspects, the comprisingmay further comprise a reporter-labeled or quencher-labeled non-naturalnucleotide.

In one embodiment, a composition is provided comprising a cleavableprobe, said probe comprising, from 5′ to 3′, (i) a first sequence regionlabeled with a fluorophore-quencher pair; (ii) a second sequence region;(iii) a sequence that is the reverse complement of the second sequenceregion; and (iv) a sequence comprising one or more ribonucleotide basethat is complimentary to a first region on a first strand of the targetnucleic acid.

In certain aspects, the composition may further comprise a polymerase,an endoribonuclease enzyme, a reference probe or free nucleotides.

In certain aspects, the cleavable probe may further comprise (v) a loopsequence of one or more nucleotides between the second sequence regionand the sequence that is the reverse complement of the second sequenceregion. In some aspects, the loop sequence may be 4-20, 6-15 or 10-15nucleotides in length. In some aspects, the loop sequence may compriseat least 3-5 consecutive A nucleotides. In some embodiments, the loopsequence comprises one or more polymerase extension blocking moieties.In certain aspects the loop sequence may comprise a combination of oneor more nucleotides and one or more extension blocking moieties.Polymerase extension blocking moieties may be used as part or all of aloop sequence. Examples of extension blocking moieties include carbonspacers. Carbon spacers may include spacers that may be 3 to 36 carbonatoms in length. Common examples of internal oligonucleotide carbonspacers include spacers that are 3, 9, and 18 carbon atoms in length.Carbon spacers may be used to prevent the cleavable probes from formingnon-specific double stranded PCR products. Carbon spacers may also beused to adjust the melt temperature (Tm) of the hairpin probe. Otherpolymerase extension blocking moieties may include non-naturalnucleotides, ribonucleotides, or any other non-nucleotide chemicalmoiety.

In a further aspect, the composition may further comprise a second (orfurther) cleavable probe as described above, wherein different probesmay be distinguishable based on having different reporters and ordifferent melt points. For example, a second (or further) probe maycomprise, from 5′ to 3′, (i) a first sequence region comprising at leasta one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a second sequence region; (iii) a sequencethat is the reverse complement of the second sequence region; and (iv) asequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of a second (orfurther) target nucleic acid. In certain aspects, the first and secondprobes may comprise distinguishable reporters and/or form hairpinshaving distinguishable melt points. In certain aspects, the cleavableprobe may further comprise (v) a loop sequence as discussed above. Insome aspects, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36 or more probes.

In some aspects, a method of the embodiments may further compriseperforming multiple polymerase chain reaction cycles. In some aspects,detecting the change in signal from the label comprises detecting thesignal before, during, or after performing the multiple polymerase chainreaction cycles. In another aspect, detecting the change in signal fromthe label comprises detecting the signal only after performing themultiple polymerase chain reaction cycles. In this aspect, the methodmay further comprise comparing the detected signal from the label to apredetermined ratio of the signal of the label to a reference signalfrom a label on a non-hybridizing probe.

In some aspects, a method of the embodiments may further comprisequantifying the amount of the target nucleic acid in the sample. Forexample, quantifying the amount of the target nucleic acid in the samplemay comprise: using a standard curve; determining a relative amount ofthe nucleic acid target; using end-point quantitation; or determining anamount of the nucleic acid target by relating the PCR cycle number atwhich the signal is detectable over background to the amount of targetpresent.

In a further embodiment, a kit is provided comprising one or more of thecompositions disclosed herein. For example, in one embodiment a kit isprovided that comprises: (a) a first cleavable probe, said probecomprising, from 5′ to 3′, (i) a first sequence region comprising atleast a one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a second sequence region; (iii) a sequencethat is the reverse complement of the second sequence region; and (iv) asequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of the target nucleicacid; and (b) a reporter-labeled non-natural nucleotide; aquencher-labeled non-natural nucleotide; or an endoribonuclease enzyme.In a further aspect, the kit comprises at least two, three, four, fiveor six probes. In some embodiments, the probes may further comprise (v)a loop sequence as discussed above. In certain aspects, the probes maycomprise distinguishable reporters or form hairpins with distinguishablemelt points. In some aspects, the kit may further comprise a polymerase,a reference probe, free nucleotides, or instructions for use of the kit.

In still a further embodiment, a method is provided for detecting thepresence of a target nucleic acid comprising: (a) contacting the samplewith a first set of probes, said set of probes comprising a cleavableprobe comprising, from 5′ to 3′, (i) a sequence region comprising atleast a one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of the target nucleic acid; and a captureprobe comprising, from 5′ to 3′, (i) a sequence region identical to thesequence region from the cleavable probe and comprising at least oneun-labeled non-natural nucleotide identical to the at least onenon-natural nucleotide from the cleavable probe; and (ii) a sequencecomplimentary to capture sequence of the cleavable probe; (b) contactingthe cleavable probe with an endoribonuclease, thereby cleaving probethat is hybridized with target nucleic acid to form a truncatedcleavable probe; (c) allowing the truncated cleavable probe to hybridizewith the capture probe; (d) extending the truncated cleavable probe inthe presence of a non-natural nucleotide labeled with a second member ofa reporter-quencher pair that is capable of base-pairing with the atleast one un-labeled non-natural nucleotide in the capture probe to forman extended cleavable probe; (e) allowing the extended cleavable probeto hybridize to itself to form a hairpin probe; and (f) detecting thetarget nucleic acid by detecting a change in signal from the label onthe cleavable probe and the hairpin probe.

In a further aspect, the method may comprise (a) contacting the samplewith a second set of probes, said set of probes comprising a cleavableprobe comprising, from 5′ to 3′, (i) a sequence region comprising atleast a one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of a second target nucleic acid; and acapture probe comprising, from 5′ to 3′, (i) a sequence region identicalto the sequence region from the cleavable probe and comprising at leastone un-labeled non-natural nucleotide identical to the at least onenon-natural nucleotide from the cleavable probe; and (ii) a sequencecomplimentary to capture sequence of the cleavable probe; (b) contactingthe cleavable probe with an endoribonuclease, thereby cleaving probethat is hybridized with the second target nucleic acid to form atruncated cleavable probe; (c) allowing the truncated cleavable probe tohybridize with the capture probe; (d) extending the truncated cleavableprobe in the presence of a quencher-labeled non-natural nucleotide thatis capable of base-pairing with the at least un-labeled one non-naturalnucleotide in the capture probe to form an extended cleavable probe; (e)allowing the extended cleavable probe to hybridize to itself to form ahairpin probe; and (f) detecting the second target nucleic acid bydetecting a change in signal from the label on the cleavable probe andthe hairpin probe.

In certain aspects, the cleavable probe may comprise a sequencecomprising 1 to 5 ribonucleotide bases that is complimentary to a firstregion on a first strand of the target nucleic acid sequence. In someaspects, the cleavable probe may comprise a sequence comprising 3 to 5ribonucleotide bases that is complimentary to a first region on a firststrand of the target nucleic acid sequence.

In yet a further aspect, the method is a multiplex method and comprises(a) contacting the sample with a third, fourth, fifth or sixth set ofprobes, said set of probes comprising a cleavable probe comprising, from5′ to 3′, (i) a sequence region comprising at least a one non-naturalnucleotide labeled with a first member of a reporter-quencher pair; (ii)a capture sequence; and (iii) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of a third, fourth, fifth or sixth target nucleic acid; and acapture probe comprising, from 5′ to 3′, (i) a sequence region identicalto the sequence region from the cleavable probe and comprising at leastone un-labeled non-natural nucleotide identical to the at least onenon-natural nucleotide from the cleavable probe; and (ii) a sequencecomplimentary to capture sequence of the cleavable probe; (b) contactingthe cleavable probe with an endoribonuclease, thereby cleaving probethat is hybridized with the third, fourth, fifth or sixth target nucleicacid to form a truncated cleavable probe; (c) allowing the truncatedcleavable probe to hybridize with the capture probe; (d) extending thetruncated cleavable probe in the presence of a quencher-labelednon-natural nucleotide that is capable of base-pairing with the at leastun-labeled one non-natural nucleotide in the capture probe to form anextended cleavable probe; (e) allowing the extended cleavable probe tohybridize to itself to form a hairpin probe; and (f) detecting thethird, fourth, fifth or sixth target nucleic acid by detecting a changein signal from the label on the cleavable probe and the hairpin probe.For example, detecting the presence of the first and/or second targetnucleic acid may be performed sequentially or essentiallysimultaneously. In still a further aspect, the first and second set ofprobes may comprise distinguishable reporters. In another aspect, thefirst and second set of probes may comprise the same reporter and, insome cases, the hairpin probes formed by the first and second probes maycomprise distinguishable melt points (e.g., melt points that differ by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C. from one another, or anyrange derivable therein). In one aspect, the hairpin probes formed bythe first, second, third, fourth, fifth, and/or sixth set of probes eachmay comprise a distinguishable label or a distinguishable melt point.Thus, in some further aspects, a multiplex method according to theembodiments can comprise the use of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 or more distinct set of probes whereineach probe comprises either (1) a distinguishable melt point or (2) adistinguishable label, such that the signal from each distinct probe maybe individually discerned.

In certain aspects, the second sequence region of the cleavable probemay comprise one or more non-natural bases. In certain aspects, after anendoribonuclease cleavage, the truncated cleavable probe and the targetnucleic acid may have a melt point of less than 55° C.

In certain aspects, the first member of a reporter-quencher pair may bea reporter, such as, for example, a fluorophore. In some aspects, thechange in the signal may be a decrease in a fluorescent signal.

In certain aspects, detecting a change in signal from the label maycomprise detecting a change in signal from a reporter as the temperatureof the sample is changed. In some aspects, detecting a change in signalfrom the reporter may comprise detecting a change in signal from thereporter as the temperature of the sample is increased above the meltpoint of hairpin probe.

In certain aspects, the cleavable probe and/or the capture probe may beattached to a solid support.

In a further embodiment, a composition is provided comprising a firstset of probes, said set of probes comprising a cleavable probecomprising, from 5′ to 3′, (i) a sequence region comprising at least aone non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of the target nucleic acid; and a captureprobe comprising, from 5′ to 3′, (i) a sequence region identical to thesequence region from the cleavable probe and comprising at least oneun-labeled non-natural nucleotide identical to the at least onenon-natural nucleotide from the cleavable probe; and (ii) a sequencecomplimentary to capture sequence of the cleavable probe. In certainaspects, the comprising may further comprise a reporter-labeled orquencher-labeled non-natural nucleotide. In certain aspects, thecomposition may comprise a polymerase, a reference probe or freenucleotides.

In a further aspect, the composition may further comprise a second setof probes comprising a cleavable probe comprising, from 5′ to 3′, (i) asequence region comprising at least a one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a capturesequence; and (iii) a sequence comprising one or more ribonucleotidebase that is complimentary to a first region on a first strand of asecond target nucleic acid; and a capture probe comprising, from 5′ to3′, (i) a sequence region identical to the sequence region from thecleavable probe and comprising at least one un-labeled non-naturalnucleotide identical to the at least one non-natural nucleotide from thecleavable probe; and (ii) a sequence complimentary to capture sequenceof the cleavable probe. In certain aspects, the first and second set ofprobes may comprise distinguishable reporters and/or form hairpinshaving distinguishable melt points. In some aspects, the compositioncomprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or moresets of probes.

In a further embodiment, a kit is provided comprising (a) a first set ofprobes, said set of probes comprising a cleavable probe comprising, from5′ to 3′, (i) a sequence region comprising at least a one non-naturalnucleotide labeled with a first member of a reporter-quencher pair; and(iii) a sequence comprising one or more ribonucleotide base that iscomplimentary to a first region on a first strand of the target nucleicacid; and a capture probe comprising, from 5′ to 3′, (i) a sequenceregion identical to the sequence region from the cleavable probe andcomprising at least one un-labeled non-natural nucleotide identical tothe at least one non-natural nucleotide from the cleavable probe; and(ii) a sequence complimentary to capture sequence of the cleavableprobe; and (b) a reporter-labeled non-natural nucleotide; aquencher-labeled non-natural nucleotide; or an endoribonuclease enzyme.In a further aspect, the kit comprises at least four sets of probes. Incertain aspects, the sets of probes may comprise distinguishablereporters or form hairpins with distinguishable melt points. In someaspects, the kit may further comprise a polymerase, a reference probe,free nucleotides, a reference sample, or instructions for use of thekit.

In yet a further embodiment, a method is provided for detecting thepresence of a target nucleic acid comprising: (a) contacting the samplewith a first set of probes, said set of probes comprising a cleavableprobe comprising, from 5′ to 3′, (i) a sequence region comprising atleast a one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of the target nucleic acid; and a captureprobe comprising, from 5′ to 3′, (i) a sequence region identical to apart of the sequence region from the cleavable probe and (ii) a sequencecomplimentary to capture sequence of the cleavable probe; (b) contactingthe cleavable probe with an endoribonuclease, thereby cleaving thecleavable probe that is hybridized with target nucleic acid to form atruncated cleavable probe; (c) allowing the truncated cleavable probe tohybridize with the capture probe; (d) extending the truncated cleavableprobe to form an extended probe; (e) allowing the extended cleavableprobe to hybridize to itself to form a hairpin probe; (f) furtherextending the hairpin probe in the presence of a non-natural nucleotidelabeled with a second member of a reporter-quencher pair that is capableof base-pairing with the at least one labeled non-natural nucleotide atthe 5′ of the cleavable probe; and (g) detecting the target nucleic acidby detecting a change in signal from the label on the cleavable probeand the hairpin probe.

In a further aspect, the method may comprise (a) contacting the samplewith a second set of probes, said set of probes comprising a cleavableprobe comprising, from 5′ to 3′, (i) a sequence region comprising atleast a one non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of a second target nucleic acid; and acapture probe comprising, from 5′ to 3′, (i) a sequence region identicalto a part of the sequence region from the cleavable probe and (ii) asequence complimentary to capture sequence of the cleavable probe; (b)contacting the cleavable probe with an endoribonuclease, therebycleaving the cleavable probe that is hybridized with target nucleic acidto form a truncated cleavable probe; (c) allowing the truncatedcleavable probe to hybridize with the capture probe; (d) extending thetruncated cleavable probe to form an extended probe; (e) allowing theextended cleavable probe to hybridize to itself to form a hairpin probe;(f) further extending the hairpin probe in the presence of a non-naturalnucleotide labeled with a second member of a reporter-quencher pair thatis capable of base-pairing with the at least one labeled non-naturalnucleotide at the 5′ of the cleavable probe; and (g) detecting thesecond target nucleic acid by detecting a change in signal from thelabel on the cleavable probe and the hairpin probe.

In certain aspects, the cleavable probe may comprise a sequencecomprising 1 to 5 ribonucleotide bases that is complimentary to a firstregion on a first strand of the target nucleic acid sequence. In someaspects, the cleavable probe may comprise a sequence comprising 3 to 5ribonucleotide bases that is complimentary to a first region on a firststrand of the target nucleic acid sequence.

In yet a further aspect, the method is a multiplex method and comprises(a) contacting the sample with a third, fourth, fifth or sixth set ofprobes, said set of probes comprising a cleavable probe comprising, from5′ to 3′, (i) a sequence region comprising at least a one non-naturalnucleotide labeled with a first member of a reporter-quencher pair; (ii)a capture sequence; and (iii) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of a third, fourth, fifth or sixth target nucleic acid; and acapture probe comprising, from 5′ to 3′, (i) a sequence region identicalto a part of the sequence region from the cleavable probe and (ii) asequence complimentary to capture sequence of the cleavable probe; (b)contacting the cleavable probe with an endoribonuclease, therebycleaving the cleavable probe that is hybridized with target nucleic acidto form a truncated cleavable probe; (c) allowing the truncatedcleavable probe to hybridize with the capture probe; (d) extending thetruncated cleavable probe to form an extended probe; (e) allowing theextended cleavable probe to hybridize to itself to form a hairpin probe;(f) further extending the hairpin probe in the presence of a non-naturalnucleotide labeled with a second member of a reporter-quencher pair thatis capable of base-pairing with the at least one labeled non-naturalnucleotide at the 5′ of the cleavable probe; and (g) detecting thethird, fourth, fifth or sixth target nucleic acid by detecting a changein signal from the label on the cleavable probe and the hairpin probe.For example, detecting the presence of the first, second and/or furthertarget nucleic acid may be performed sequentially or essentiallysimultaneously. In still a further aspect, first, second and/or furtherset of probes may comprise distinguishable reporters. In another aspect,first, second and/or further set of probes may comprise the samereporter and, in some cases, the hairpin probes formed by the first andsecond probes may comprise distinguishable melt points (e.g., meltpoints that differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30° C. fromone another, or any range derivable therein). In one aspect, the hairpinprobes formed by the first, second, third, fourth, fifth, and/or sixthset of probes each may comprise a distinguishable label or adistinguishable melt point. Thus, in some further aspects, a multiplexmethod according to the embodiments can comprise the use of at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more distinctset of probes wherein each probe comprises either (1) a distinguishablemelt point or (2) a distinguishable label, such that the signal fromeach distinct probe may be individually discerned.

In certain aspects, the second sequence region of the cleavable probemay comprise one or more non-natural bases. In certain aspects, after anendoribonuclease cleavage, the truncated cleavable probe and the targetnucleic acid may have a melt point of less than 55° C.

In certain aspects, the first member of a reporter-quencher pair may bea reporter, such as, for example, a fluorophore. In some aspects, thechange in the signal may be a decrease in a fluorescent signal.

In certain aspects, detecting a change in signal from the label maycomprise detecting a change in signal from a reporter as the temperatureof the sample is changed. In some aspects, detecting a change in signalfrom the reporter may comprise detecting a change in signal from thereporter as the temperature of the sample is increased above the meltpoint of hairpin probe.

In certain aspects, the cleavable probe and/or the capture probe may beattached to a solid support.

In a further embodiment, a composition is provided comprising a firstset of probes, said set of probes comprising a cleavable probecomprising, from 5′ to 3′, (i) a sequence region comprising at least aone non-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of the target nucleic acid; and a captureprobe comprising, from 5′ to 3′, (i) a sequence region identical to apart of the sequence region from the cleavable probe and (ii) a sequencecomplimentary to capture sequence of the cleavable probe. In certainaspects, the comprising may further comprise a reporter-labeled orquencher-labeled non-natural nucleotide. In certain aspects, thecomposition may comprise a polymerase, a reference probe or freenucleotides.

In a further aspect, the composition may further comprise a second setof probes, said set of probes comprising a cleavable probe comprising,from 5′ to 3′, (i) a sequence region comprising at least a onenon-natural nucleotide labeled with a first member of areporter-quencher pair; (ii) a capture sequence; and (iii) a sequencecomprising one or more ribonucleotide base that is complimentary to afirst region on a first strand of a second target nucleic acid; and acapture probe comprising, from 5′ to 3′, (i) a sequence region identicalto a part of the sequence region from the cleavable probe and (ii) asequence complimentary to capture sequence of the cleavable probe. Incertain aspects, the first and second set of probes may comprisedistinguishable reporters and/or form hairpins having distinguishablemelt points. In some aspects, the composition comprises 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more sets of probes.

In a further embodiment, a kit is provided comprising (a) a first set ofprobes, said set of probes comprising a cleavable probe comprising, from5′ to 3′, (i) a sequence region comprising at least a one non-naturalnucleotide labeled with a first member of a reporter-quencher pair; (ii)a capture sequence; and (iii) a sequence comprising one or moreribonucleotide base that is complimentary to a first region on a firststrand of the target nucleic acid; and a capture probe comprising, from5′ to 3′, (i) a sequence region identical to a part of the sequenceregion from the cleavable probe and (ii) a sequence complimentary tocapture sequence of the cleavable probe; and (b) a reporter-labelednon-natural nucleotide; a quencher-labeled non-natural nucleotide; or anendoribonuclease enzyme. In a further aspect, the kit comprises at leastfour sets of probes. In certain aspects, the sets of probes may comprisedistinguishable reporters or form hairpins with distinguishable meltpoints. In some aspects, the kit may further comprise a polymerase, areference probe, free nucleotides, a reference sample, or instructionsfor use of the kit.

In another embodiment, a method is provided for detecting the presenceof a target nucleic acid comprising: (a) contacting the sample with afirst cleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising at least one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence that is complimentary to a firstregion on a first strand of the target nucleic acid; (b) hybridizing thecleavable probe and an upstream primer to the target nucleic acid, andperforming extension using a polymerase possessing 5′ nuclease activity;(c) extending the nucleic acid sequence until contacting the cleavablehairpin probe with a polymerase possessing nuclease activity, therebycleaving the probe that is hybridized with target nucleic acid to form atruncated cleavable probe; (d) allowing the truncated cleavable probe tohybridize to itself to form a hairpin probe; (e) extending the hairpinprobe in the presence of a non-natural nucleotide labeled with a secondmember of a reporter-quencher pair that is capable of base-pairing withthe at least one non-natural nucleotide of the first sequence region;and (f) detecting the target nucleic acid by detecting a change insignal from the label on the hairpin probe. In certain aspects, aportion of the sequence that is the reverse complement of the secondsequence region (iii) may be complimentary to a first region on a firststrand of the target nucleic acid. In certain embodiments, the methodmay further comprise performing melt analysis on the hairpin probe.

In another embodiment, a method is provided for detecting the presenceof a target nucleic acid comprising: (a) contacting the sample with afirst cleavable hairpin probe, said probe comprising, from 5′ to 3′, (i)a first sequence region labeled with a reporter-quencher pair; (ii) asecond sequence region; (iii) a sequence that is the reverse complementof the second sequence region; and (iv) a sequence that is complimentaryto a first region on a first strand of the target nucleic acid; (b)hybridizing the cleavable probe and an upstream primer to the targetnucleic acid, and performing extension using a polymerase possessing 5′nuclease activity; (c) extending the nucleic acid sequence untilcontacting the cleavable hairpin probe with a polymerase possessingnuclease activity, thereby cleaving the probe that is hybridized withtarget nucleic acid to form a truncated cleavable probe; (d) allowingthe truncated probe to hybridize to itself to form a cleaved hairpinprobe; (e) extending the cleaved hairpin probe onto itself such that theflurophore and quencher are physically separated; (f) detecting thetarget nucleic acid by detecting a change in signal from the extendedhairpin probe.

Certain aspects of the embodiments concern the use of at least onenon-natural nucleotide. In some aspects, the non-natural nucleotide isan isobase, such as iso-guanine (isoG) or iso-cytosine (isoC). In thisaspect, the quencher-labeled non-natural nucleotide is a cognate isoC(or isoG). In still further aspects, at least one of the first and/orsecond primers comprises at least one non-natural nucleotide in thetarget-specific sequence. For example, in some aspects, the non-naturalnucleotide in the target-specific sequence regulates sequence-specificannealing thereby enhancing primer-template hybridization forsequence-specific amplification of nucleotides (see, e.g., PCT Publn.WO/2011/050278, incorporated herein by reference).

The cleavage and extension of the cleavable probes as disclosed hereinmay be performed under isothermal conditions in which the cleavableprobes are cleaved and extended while reaction conditions are maintainedat a substantially constant temperature. Isothermal amplification ofsignal may be achieved because both fragments of a cleaved probe possessa lower melting temperature than the probe to target before cleavage.This causes the two fragments to disassociate from the target, allowinganother probe to hybridize and cleave. This process repeats itselfallowing multiple probes to cleave and extend from a single target at aconstant temperature. This feature is unique compared to other methodsrelated to closed tube multiplexed detection by melt analysis, whichrely on 5′-nuclease activity to obtain unique melt signatures, whichcannot amplify the signal of targets or amplicons isothermally.Alternatively, the cleavage and extension of the cleavable probes asdisclosed herein may be performed under non-isothermal conditions, suchas under the cycling temperature conditions of PCR.

In some aspects, a method of the embodiments may further compriseperforming an amplification step to amplify a target sequence. Thecleavage and extension of the cleavable probes may be performed duringor subsequent to the amplification process. For example, theamplification can be isothermal amplification or one or more polymerasechain reaction cycles. Isothermal amplification techniques include, forexample, strand displacement amplification (SDA), loop-mediatedamplification (LAMP), rolling circle amplification (RCA), andhelicase-dependent amplification (HAD) (see, e.g., Yan et al., 2014). Insome aspects, detecting the change in signal from the label comprisesdetecting the signal before, during, or after performing the isothermalamplificiation or the multiple polymerase chain reaction cycles. Inanother aspect, detecting the change in signal from the label comprisesdetecting the signal only after performing the isothermal amplificiationor the multiple polymerase chain reaction cycles. In this aspect, themethod may further comprise comparing the detected signal from the labelto a predetermined ratio of the signal of the label to a referencesignal from a label on a non-hybridizing probe.

In some aspects, a method of the embodiments may further comprisequantifying the amount of the target nucleic acid in the sample. Forexample, quantifying the amount of the target nucleic acid in the samplemay comprise: using digital PCR; using a standard curve; determining arelative amount of the nucleic acid target; using end-pointquantitation; or determining an amount of the nucleic acid target byrelating the PCR cycle number at which the signal is detectable overbackground to the amount of target present.

In various aspects of the present methods, detecting a change in signalfrom the reporter may comprise detecting the change (or rate of change)in signal, such as unquenching of a signal as the temperature of thesample is changed. In one aspect, the temperature of the sample may beincreased above (or decreased below) the melt point of the hairpin ofone more of the primers in the sample. In the case where two or moreprimer sets are present, changing the temperature of a sample maycomprise increasing the temperature of the sample from a temperaturethat is below the melt point of the hairpins of both of the firstprimers in the first and second set of primers to a temperature that isabove the melt point of both of the hairpins.

In various aspects, the probes of the embodiments may comprise the samereporter and comprise hairpins with distinguishable melt points (e.g.,melt points that differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C. from one another, or any range derivable therein).

In further various aspects, a multiplex method according to theembodiments can comprise the use of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 or more distinct probe sets wherein eachprobe or probe set comprises either (1) a hairpin with a distinguishablemelt point or (2) a distinguishable reporter, such that the signal fromeach distinct probe or probe set may be individually discerned.

In a further embodiment, a kit is provided comprising one or more probesor probe sets of the embodiments. In a further aspect, the kit furthercomprises a polymerase with exonuclease activity, an endoribonuclease(e.g., an RNase H), a reference probe, free nucleotides, freenon-natural nucleotide, a reference sample and/or instructions for useof the kit.

As used herein a solid support may be beads with magnetic propertiesand/or beads with a density that allows them to rest upon a twodimensional surface in solution. The particles may in one way or anotherrest upon a two dimensional surface by magnetic, gravitational, or ionicforces, or by chemical bonding, or by any other means known to thoseskilled in the art. Particles may consist of glass, polystyrene, latex,metal, quantum dot, polymers, silica, metal oxides, ceramics, or anyother substance suitable for binding to nucleic acids, or chemicals orproteins which can then attach to nucleic acids. The particles may berod shaped or spherical or disc shaped, or comprise any other shape. Theparticles may also be distinguishable by their shape or size or physicallocation. The particles may be spectrally distinct by virtue of having acomposition containing dyes or ratios or concentrations of one or moredyes or fluorochromes, or may be distinguishable by barcode orholographic images or other imprinted forms of particle coding. Wherethe particles are magnetic particles, they may be attracted to thesurface of the chamber by application of a magnetic field. Likewise,magnetic particles may be dispersed from the surface of the chamber byremoval of the magnetic field. The magnetic particles are preferablyparamagnetic or superparamagnetic. Paramagnetic and superparamagneticparticles have negligible magnetism in the absence of a magnetic field,but application of a magnetic field induces alignment of the magneticdomains in the particles, resulting in attraction of the particles tothe field source. When the field is removed, the magnetic domains returnto a random orientation so there is no interparticle magnetic attractionor repulsion. In the case of superparamagnetism, this return to randomorientation of the domains is nearly instantaneous, while paramagneticmaterials will retain domain alignment for some period of time afterremoval of the magnetic field. Where the particles have a sufficientdensity they may be attracted to the bottom surface of the chamber bygravity, and dispersed from the bottom surface of the chamber byagitation of the chamber, such as by vortexing, sonication, or fluidicmovement. Agitation of the chamber may also be used to further assist indispersing particles in methods and systems in which the particles wereattracted to a surface of the chamber by other forces, such as magneticor ionic forces, or suction forces, or vacuum filtration, or affinity,or hydrophilicity or hydrophobicity, or any combination thereof.

A reporter or labeling agent, is a molecule that facilitates thedetection of a molecule (e.g., a nucleic acid sequence) to which it isattached. Numerous reporter molecules that may be used to label nucleicacids are known. Direct reporter molecules include fluorophores,chromophores, and radiophores. Non-limiting examples of fluorophoresinclude, a red fluorescent squarine dye such as2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dio-xolate,an infrared dye such as 2,4Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,-3-dioxolate,or an orange fluorescent squarine dye such as2,4-Bis[3,5-dimethyl-2-pyrrolyl]cyclobutenediylium-1,3-diololate.Additional non-limiting examples of fluorophores include quantum dots,Alexa Fluor™ dyes, AMCA, BODIPY™ 630/650, BODIPY™ 650/665, BODIPY™-FL,BODIPY™-R6G, BODIPY™-TMR, BODIPY™-TRX, Cascade Blue™, CyDye™, includingbut not limited to Cy2™, Cy3™, and Cy5™, a DNA intercalating dye,6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green™ 488, Oregon Green™ 500,Oregon Green™ 514, Pacific Blue™, REG, phycobilliproteins including, butnot limited to, phycoerythrin and allophycocyanin, Rhodamine Green™,Rhodamine Red™, ROX™, TAMRA™, TET™, Tetramethylrhodamine, or Texas Red™.A signal amplification reagent, such as tyramide (PerkinElmer), may beused to enhance the fluorescence signal. Indirect reporter moleculesinclude biotin, which must be bound to another molecule such asstreptavidin-phycoerythrin for detection. Pairs of labels, such asfluorescence resonance energy transfer pairs or dye-quencher pairs, mayalso be employed.

Labeled amplification products may be labeled directly or indirectly.Direct labeling may be achieved by, for example, using labeled primers,using labeled dNTPs, using labeled nucleic acid intercalating agents, orcombinations of the above. Indirect labeling may be achieved by, forexample, hybridizing a labeled probe to the amplification product.

The methods disclosed herein may further comprise quantifying theinitial amount of the nucleic acid target(s) in the sample. Thequantification may comprise, for example, determining the relativeconcentrations of DNA present during the exponential phase of thereal-time PCR by plotting fluorescence against cycle number on alogarithmic scale. The amounts of DNA may then be determined bycomparing the results to a standard curve produced by real-time PCR ofserial dilutions of a known amount of DNA. Additionally, real-time PCRmay be combined with reverse transcription polymerase chain reaction toquantify RNAs in a sample, including low abundance RNAs. Alternatively,quantification may be accomplished by digital PCR.

The target nucleic acid sequence may be any sequence of interest. Thesample containing the target nucleic acid sequence may be any samplethat contains nucleic acids. In certain aspects of the invention thesample is, for example, a subject who is being screened for the presenceor absence of one or more genetic mutations or polymorphisms. In anotheraspect of the invention the sample may be from a subject who is beingtested for the presence or absence of a pathogen. Where the sample isobtained from a subject, it may be obtained by methods known to those inthe art such as aspiration, biopsy, swabbing, venipuncture, spinal tap,fecal sample, or urine sample. In some aspects of the invention, thesample is an environmental sample such as a water, soil, or air sample.In other aspects of the invention, the sample is from a plant, bacteria,virus, fungi, protozoan, or metazoan.

Each amplification cycle has three phases: a denaturing phase, a primerannealing phase, and a primer extension phase. The amplification cyclecan be repeated until the desired amount of amplification product isproduced. Typically, the amplification cycle is repeated between about10 to 40 times. For real-time PCR, detection of the amplificationproducts will typically be done after each amplification cycle. Althoughin certain aspects of the invention, detection of the amplificationproducts may be done after every second, third, fourth, or fifthamplification cycle. Detection may also be done such that as few as 2 ormore amplification cycles are analyzed or detected. The amplificationcycle may be performed in the same chamber in which the detection of theamplification occurs, in which case this chamber would need to comprisea heating element so the temperature in the chamber can be adjusted forthe denaturing phase, primer annealing phase, and a primer extensionphase of the amplification cycle. The heating element would typically beunder the control of a processor. The amplification cycle may, however,be performed in a different chamber from the chamber in which detectionof the amplification occurs, in which case the “amplification” chamberwould need to comprise a heating element but the “detection” or“imaging” chamber would not be required to have a heating element. Whereamplification and detection occur in separate chambers, the fluid inwhich the amplification reaction occurs may be transferred between thechambers by, for example, a pump or piston. The pump or piston may beunder the control of a processor. Alternatively, the fluid may betransferred between the chambers manually using, for example, a pipette.

Amplification may be performed in a reaction mixture that includes atleast one non-natural nucleotide having a non-natural nucleotide. The atleast one non-natural nucleotide of the reaction mixture may base pairwith the at least one non-natural nucleotide present in the primer ofthe first and/or second primer set. Optionally, the non-naturalnucleotide is coupled to a label which may include fluorophores andquenchers. The quencher may quench a fluorophore present in the primerof the first and/or second primer set.

Detecting may include amplifying one or more polynucleotides of thepopulation. For example, detecting may include amplifying one or morepolynucleotides of the population in the presence of at least onenon-natural nucleotide. The non-natural nucleotide may have anon-natural nucleotide (e.g., isoC and isoG), which, optionally, iscapable of base-pairing with the non-natural nucleotide of the mixtureof oligonucleotides (e.g., a non-natural nucleotide present in thedegenerate oligonucleotides). The non-natural nucleotide may be coupledto a label. Suitable labels include fluorophores and quenchers.

The method may be used to detect the target continuously duringamplification or in real-time. The method may be used quantitatively.

Certain aspects of the embodiments concern endoribonuclease enzymes anduse of such enzymes to specifically cleave probes having aribonucleotide (RNA) position when the probe is hybridized to a DNAtarget sequence. In some aspects, the endoribonuclease is an RNAse H,such as RNase HII. In certain specific aspects, the endoribonuclease isa thermostable enzyme or a thermophilic, hotstart enzyme (e.g., athermostable RNase HII enzyme and a thermophilic, hotstart RNaseHIIenzyme).

Amplification may be performed in the presence of one or morenon-natural nucleotides and/or in the presence of at least one quenchercoupled to a non-natural nucleotide. In some embodiments, thenon-natural nucleotide coupled to the at least one quencher may beisoCTP or isoGTP.

In some methods, the first and second labels may be different. In somemethods the first and second quencher may be different and may becapable of quenching two different fluorophores. In other methods, thefirst and second quenchers may be the same and may be capable ofquenching two different fluorophores.

The methods described herein may include determining a meltingtemperature for an amplicon (e.g., amplified nucleic acid of at leastone of amplified nucleic acid of HIV and amplified control nucleicacid). The methods may include determining a melting temperature for anucleic acid complex that includes a labeled probe hybridized to atarget nucleic acid (which may include amplified target nucleic acid).The melting temperature may be determined by exposing the amplicon ornucleic acid complex to a gradient of temperatures and observing asignal from a label. Optionally, the melting temperature may bedetermined by (a) reacting an amplicon with an intercalating agent at agradient of temperatures and (b) observing a detectable signal from theintercalating agent. The melting temperature of a nucleic acid complexmay be determined by (1) hybridizing a probe to a target nucleic acid toform a nucleic acid complex, where at least one of the probe and thetarget nucleic acid includes a label; (2) exposing the nucleic acidcomplex to a gradient of temperatures; and (3) observing a signal fromthe label.

The methods may be performed in any suitable reaction chamber under anysuitable conditions. For example, the methods may be performed in areaction chamber without opening the reaction chamber. The reactionchamber may be part of an array of reaction chambers. In someembodiments, the steps of the methods may be performed separately indifferent reaction chambers.

The methods disclosed herein may be performed in droplets. Likewise, thecompositions disclosed herein may be disposed within droplets. Forexample, the cleavable probes disclosed herein may be divided into manyseparate reactions for PCR or isothermal amplification using droplets.Thus, in certain embodiments the methods disclosed herein arecompartmentalized in droplets to perform quantitative digital PCRreactions, or other quanitative digital amplification reactions. Asdescribed in Vogelstein et al., 1999, at pgs. 9236-9241, digital PCRmethods may be helpful for distributing the target nucleic acid suchthat the vast majority of reactions contain either one or zero targetnucleic acid molecules. At certain dilutions the number of amplificationpositive reactions is equal to the number of template moleculesoriginally present.

In some embodiments, the methods may be capable of detecting no morethan about 100 copies of the target nucleic acid in a sample (e.g., in asample having a volume of about 25 microliters). In other embodiments,the methods may be capable of detecting no more than about 500 copies,1000 copies, 5000 copies, or 10,000 copies in a sample (e.g., in asample having a volume of about 25 microliters).

In other embodiments, the methods may be capable of detecting no morethan about 100 copies of target nucleic acid in a sample (e.g., in asample having a volume of about 25 microliters) using real-timedetection in no more than about 150 cycles of the PCR, no more thanabout 100 cycles, no more than about 90 cycles, no more than about 80cycles, no more than about 70 cycles, no more than about 60 cycles, nomore than about 50 cycles, no more than about 40 cycles, or no more thanabout 30 cycles of the PCR.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-B—A non-limiting exemplary schematic showing a probe system ofthe embodiments. FIG. 1A, The cleavable probe comprises areporter-labeled isoG nucleotide (“isoG*”) at its 5′ end, a firstsequence region (“Tag A”), a second sequence region (“Tag B”), a loopsequence, a sequence region that is the reverse compliment of Tag B(“Tag B complement”); and a sequence complementary to the targetamplicon (indicated as “A”). The cleavable probe also comprises one ormore ribonucleotides (indicated by the solid square) in the “A” sequenceand may comprise a modification that blocks extension on the 3′ end(indicated as “P”). In the presence of a target amplicon the cleavableprobe hybridizes to the amplicon and is cleaved at the ribonucleotideposition by RNase H. Following cleavage, the probe can hybridize toitself via the Tag B and Tag B complement sequences to form a hairpin.Extension of the probe will synthesize sequences complementary to theTag A sequences and will incorporate a quencher labeled isoC(“isoC^(Q)”). The resulting hairpin probe quenches the fluorescence ofthe labeled isoG. FIG. 1B, The probes can be designed to have uniquemelt temperatures (T_(m)), such as by adjusting the sequence and lengthof the sequence regions. Thus, a melt analysis can be performed todifferentiation probes having different melt temperatures (and thusunquenching at different temperatures).

FIG. 2—Non-limiting exemplary probe constructs of the embodiments withvariable stem, loop, T_(m) and delta G. The probes were designed asdetailed in FIG. 1. The sequences of each probe are shown (SEQ ID NOS:1-11 as listed from top to bottom). Tag A sequences are in bold and thestem is comprised of 3 segments: sequence specific (B, underlinednucleotides), universal sequence (C, italicized nucleotides) and anextendable universal sequence (A, bold font nucleotides) ending with afluorophore-labelled isobase.

FIG. 3—Non-limiting exemplary target-specific probe designs of theembodiments (SEQ ID NOS: 12-21 as listed from top to bottom). The threesegments of the stem are illustrated as in FIG. 2.

FIG. 4—Graph shows the temperature gradient used to assess the hairpinfolding of the constructs shown in FIG. 2.

FIG. 5—Graph shows fluorescence quenching as a function of time for theRTx-5 construct as the annealing temperature is stepped down (see, e.g.,FIG. 4). Complete quenching was observed by the 71° C. temperature step.

FIG. 6—Graph shows fluorescence quenching as a function of time for theRTx-10 construct as the annealing temperature is stepped down (see,e.g., FIG. 4). Complete quenching was observed by the 62° C. temperaturestep.

FIG. 7—Graph shows fluorescence quenching as a function of time for theRTx-11 construct as the annealing temperature is stepped down (see,e.g., FIG. 4). Complete quenching was observed by the 41° C. temperaturestep.

FIGS. 8A-8C—Graphs show amplification (upper panels) and melt curves(lower panels) obtained from constructs RTx-1 and RTx-2 at 50° C., 62°C. and 68° C.

FIGS. 9A-9C—Graphs show amplification (upper panels) and melt curves(lower panels) obtained from constructs RTx-7 and RTx-8 at 50° C., 62°C. and 68° C.

FIGS. 10A-10C—Graphs show amplification (upper panels) and melt curves(lower panels) obtained from constructs RTx-9, RTx-10, and RTx-11 at 50°C., 62° C. and 68° C.

FIGS. 11A-11D—Graphs show amplification (upper panels) and melt (lowerpanels) curves of full length probes FL-RTx-2-20 (A), FL-RTx-2-12AT1(B), FL-RTx-2c (C), and FL-RTx-2-12-AT-4 (D). Controls: water=thin solidline, clinical negative specimen=dashed line. The test probe results areshown in thick solid lines.

FIG. 12—A non-limiting exemplary schematic showing a probe system of theembodiments. The reporter probe comprises a reporter-labeled isoCnucleotide (“isoC*”) at its 5′ end, a first sequence region (“region1”), a sequence that includes isoG and/or isoC positions (the“isoprimer”); and a sequence complementary to the amplicon (indicated as“A”). The sequence that is complementary to the amplicon also includesat least one ribonucleotide position. In the presence of a targetamplicon the reporter probe hybridizes to the amplicon and is cleaved atthe ribonucleotide position by RNase H. Following cleavage, the reporterprobe can hybridize to a capture oligonucleotide (“capture oligo”),which comprises a capture segment complimentary to the isoprimer and,optionally, a portion that “A” sequence, followed by a mirror tag regionand a 3′ unlabeled isoC. Extension of the reporter probe will synthesizesequences complementary to the mirror region 1 on the capture oligo andwill incorporate a quencher labeled isoG (“isoG^(Q)”). The extendedreporter probe now includes a region 1 and region 1 complement sequence,which allows the probe to form a hairpin and thereby quench thefluorescence of the labeled isoC. The probes can be designed to haveunique melt temperatures (T_(m)), such as by adjusting the sequence andlength of the first sequence region. Thus, a melt analysis can beperformed to differentiation probes having different melt temperatures(and thus unquenching at different temperatures).

FIG. 13—A graph of the inverted derivative of data obtained during meltanalysis.

FIG. 14—Melt profile data for multiplex probes using the samefluorophore.

FIG. 15—A non-limiting exemplary schematic showing a probe system of theembodiments in which the probe comprises both a fluorophore (“F”) and aquencher (“Q”), and ribocleavage (“R”) site. Following cleavage at theribocleavage site, extension results in separation of the fluorophoreand quencher such that a detectable change in the signal can beobserved.

FIG. 16—A non-limiting exemplary schematic showing a probe system of theembodiments in which the probe comprises both a fluorophore (“F”) and aquencher (“Q”). 5′ nuclease cleavage followed by extension results inseparation of the fluorophore and quencher such that a detectable changein the signal can be observed.

FIG. 17—A non-limiting exemplary schematic showing a probe system of theembodiments in which the probe comprises a loop sequence of one or morenucleotides located between the second sequence region and the sequencethat is the reverse complement of the second sequence region (B and B′),wherein the loop sequence is complimentary to a sequence of the targetnucleic acid.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Melt analysis assays utilize melt or anneal peaks to discriminateamplicon identity, but these melt peaks are not easily distinguishablein amplicons that melt near the same temperature and are subject to thenatural sequence composition of the target. By creating hairpinsequences with unique melt profiles, multiplexing can be achieved in asingle color channel, thus allowing even more multiplexing with multiplecolor channels.

Disclosed are methods and kits for detecting nucleic acids in a sample.Typically, the methods include detecting signals, such as a signalemitted from a fluorophore. Also disclosed are oligonucleotides,especially probes, which may be used for the detection of target nucleicacids. In particular methods of the embodiments employ an extendableprobe to facilitate multiplexing by generation of multiple melt curvesper fluorophore. In some cases, the probe is comprised of a hairpinstructure with a sequence-specific tail at the 3′-end and an extendableuniversal sequence at the 5′ end terminating in a fluorophore labelledisobase. Unlike other probe based chemistries, the sequence specificsegment is used for target identification and the release of the hairpinfor detection. In some aspects, the release of the hairpin is based oncleavage of RNA/DNA hybrid created as the sequences specific tail of theprobe hybridizes to the template. Thus, none or only a few (e.g., 3-4)bases of the sequence-specific segment are incorporated into the hairpinstructure, which is mainly comprised of target independent sequences.Varying the length of the extendable segment of the hairpin gives riseto hairpins with various sizes allowing for generation of multiple meltcurves per fluorophore.

I. Definitions

As used herein “nucleic acid” means either DNA or RNA, single-strandedor double-stranded, and any chemical modifications thereof.Modifications include, but are not limited to, those which provide otherchemical groups that incorporate additional charge, polarizability,hydrogen bonding, electrostatic interaction, and fluxionality to thenucleic acid ligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbonemodifications, methylations, and unusual base-pairing combinations, suchas the isobases. Accordingly, the nucleic acids described herein includenot only the standard bases adenine (A), cytosine (C), guanine (G),thymine (T), and uracil (U) but also non-standard or non-naturalnucleotides. Non-standard or non-natural nucleotides, which formhydrogen-bonding base pairs, are described, for example, in U.S. Pat.Nos. 5,432,272, 5,965,364, 6,001,983, 6,037,120, and 6,140,496, all ofwhich are incorporated herein by reference. By “non-standard nucleotide”or “non-natural nucleotide” it is meant a base other than A, G, C, T, orU that is susceptible to incorporation into an oligonucleotide and thatis capable of base-pairing by hydrogen bonding, or by hydrophobic,entropic, or van der Waals interactions, with a complementarynon-standard or non-natural nucleotide to form a base pair. Someexamples include the base pair combinations of iso-C/iso-G, K/X, K/P,H/J, and M/N, as illustrated in U.S. Pat. No. 6,037,120, incorporatedherein by reference.

The hydrogen bonding of these non-standard or non-natural nucleotidepairs is similar to those of the natural bases where two or threehydrogen bonds are formed between hydrogen bond acceptors and hydrogenbond donors of the pairing non-standard or non-natural nucleotides. Oneof the differences between the natural bases and these non-standard ornon-natural nucleotides is the number and position of hydrogen bondacceptors and hydrogen bond donors. For example, cytosine can beconsidered a donor/acceptor/acceptor base with guanine being thecomplementary acceptor/donor/donor base. Iso-C is anacceptor/acceptor/donor base and iso-G is the complementarydonor/donor/acceptor base, as illustrated in U.S. Pat. No. 6,037,120,incorporated herein by reference.

Other non-natural nucleotides for use in oligonucleotides include, forexample, naphthalene, phenanthrene, and pyrene derivatives as discussed,for example, in Ren, et al., 1996 and McMinn et al., 1999, both of whichare incorporated herein by reference. These bases do not utilizehydrogen bonding for stabilization, but instead rely on hydrophobic orvan der Waals interactions to form base pairs.

As used herein, the term “sample” is used in its broadest sense. Asample may include a bodily tissue or a bodily fluid including but notlimited to blood (or a fraction of blood, such as plasma or serum),lymph, mucus, tears, urine, and saliva. A sample may include an extractfrom a cell, a chromosome, organelle, or a virus. A sample may compriseDNA (e.g., genomic DNA), RNA (e.g., mRNA), and/or cDNA, any of which maybe amplified to provide an amplified nucleic acid. A sample may includenucleic acid in solution or bound to a substrate (e.g., as part of amicroarray). A sample may comprise material obtained from anenvironmental locus (e.g., a body of water, soil, and the like) ormaterial obtained from a fomite (i.e., an inanimate object that servesto transfer pathogens from one host to another).

The term “source of nucleic acid” refers to any sample that containsnucleic acids (RNA or DNA). Particularly preferred sources of targetnucleic acids are biological samples including, but not limited to,blood, plasma, serum, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum, and semen.

As used herein, the term “limit of detection” refers to the lowest levelor amount of an analyte, such as a nucleic acid, that can be detectedand quantified. Limits of detection can be represented as molar values(e.g., 2.0 nM limit of detection), as gram measured values (e.g., 2.0microgram limit of detection under, for example, specified reactionconditions), copy number (e.g., 1×10⁵ copy number limit of detection),or other representations known in the art.

As used herein the term “isolated” in reference to a nucleic acidmolecule refers to a nucleic acid molecule that is separated from theorganisms and biological materials (e.g., blood, cells, serum, plasma,saliva, urine, stool, sputum, nasopharyngeal aspirates and so forth)that are present in the natural source of the nucleic acid molecule. Anisolated nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In someembodiments, nucleic acid molecules encoding polypeptides/proteins mayalso be isolated or purified. Methods of nucleic acid isolation are wellknown in the art and may include total nucleic acidisolation/purification methods, RNA-specific isolation/purificationmethods, or DNA-specific isolation/purification methods.

As used herein, the term “microarray” refers to an arrangement of aplurality of polynucleotides, polypeptides, or other chemical compoundson a substrate. The terms “element” and “array element” refer to apolynucleotide, polypeptide, or other chemical compound having a uniqueand defined position on a microarray.

As used herein, an oligonucleotide is understood to be a molecule thathas a sequence of bases on a backbone comprised mainly of identicalmonomer units at defined intervals. The bases are arranged on thebackbone in such a way that they can enter into a bond with a nucleicacid having a sequence of bases that are complementary to the bases ofthe oligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. A distinction may be made betweenoligodeoxyribonucleotides, made up of “dNTPs,” which do not have ahydroxyl group at the 2′ position, and oligoribonucleotides, made up of“NTPs,” which have a hydroxyl group in the 2′ position. Oligonucleotidesalso may include derivatives, in which the hydrogen of the hydroxylgroup is replaced with an organic group, e.g., an allyl group.

An oligonucleotide is a nucleic acid that includes at least twonucleotides. Oligonucleotides used in the methods disclosed hereintypically include at least about ten (10) nucleotides and more typicallyat least about fifteen (15) nucleotides. Preferred oligonucleotides forthe methods disclosed herein include about 10-25 nucleotides. Anoligonucleotide may be designed to function as a “primer.” A “primer” isa short nucleic acid, usually a ssDNA oligonucleotide, which may beannealed to a target polynucleotide by complementary base-pairing. Theprimer may then be extended along the target DNA or RNA strand by apolymerase enzyme, such as a DNA polymerase enzyme. Primer pairs can beused for amplification (and identification) of a nucleic acid sequence(e.g., by the polymerase chain reaction (PCR)). An oligonucleotide maybe designed to function as a “probe.” A “probe” refers to anoligonucleotide, its complements, or fragments thereof, which are usedto detect identical, allelic, or related nucleic acid sequences. Probesmay include oligonucleotides that have been attached to a detectablelabel or reporter molecule. Typical labels include fluorescent dyes,quenchers, radioactive isotopes, ligands, scintillation agents,chemiluminescent agents, and enzymes.

An oligonucleotide may be designed to be specific for a target nucleicacid sequence in a sample. For example, an oligonucleotide may bedesigned to include “antisense” nucleic acid sequence of the targetnucleic acid. As used herein, the term “antisense” refers to anycomposition capable of base-pairing with the “sense” (coding) strand ofa specific target nucleic acid sequence. An antisense nucleic acidsequence may be “complementary” to a target nucleic acid sequence. Asused herein, “complementarity” describes the relationship between twosingle-stranded nucleic acid sequences that anneal by base-pairing. Forexample, 5′-AGT-3′ pairs with its complement, 3′-TCA-5′. In someembodiments, primers or probes may be designed to include mismatches atvarious positions. As used herein, a “mismatch” means a nucleotide pairthat does not include the standard Watson-Crick base pairs, ornucleotide pairs that do not preferentially form hydrogen bonds. Themismatch may include a natural nucleotide or a non-natural ornon-standard nucleotide substituted across from a particular base orbases in a target. For example, the probe or primer sequence 5′-AGT-3′has a single mismatch with the target sequence 3′-ACA-5′. The 5′ “A” ofthe probe or primer is mismatched with the 3′ “A” of the target.Similarly, the target sequence 5′-AGA-3′ has a single mismatch with theprobe or primer sequence 3′-(iC)CT-5′. Here an iso-C is substituted inplace of the natural “T.” However, the sequence 3′-(iC)CT-5′ is notmismatched with the sequence 5′-(iG)GA-3′.

Oligonucleotides may also be designed as degenerate oligonucleotides. Asused herein, “degenerate oligonucleotide” is meant to include apopulation, pool, or plurality of oligonucleotides comprising a mixtureof different sequences where the sequence differences occur at aspecified position in each oligonucleotide of the population. Varioussubstitutions may include any natural or non-natural nucleotide, and mayinclude any number of different possible nucleotides at any givenposition. For example, the above degenerate oligonucleotide may insteadinclude R=iC or iG, or R=A or G or T or C or iC or iG.

Oligonucleotides, as described herein, typically are capable of forminghydrogen bonds with oligonucleotides having a complementary basesequence. These bases may include the natural bases, such as A, G, C, T,and U, as well as artificial, non-standard or non-natural nucleotidessuch as iso-cytosine and iso-guanine. As described herein, a firstsequence of an oligonucleotide is described as being 100% complementarywith a second sequence of an oligonucleotide when the consecutive basesof the first sequence (read 5′-to-3′) follow the Watson-Crick rule ofbase pairing as compared to the consecutive bases of the second sequence(read 3′-to-5′). An oligonucleotide may include nucleotidesubstitutions. For example, an artificial base may be used in place of anatural base such that the artificial base exhibits a specificinteraction that is similar to the natural base.

An oligonucleotide that is specific for a target nucleic acid also maybe specific for a nucleic acid sequence that has “homology” to thetarget nucleic acid sequence. As used herein, “homology” refers tosequence similarity or, interchangeably, sequence identity, between twoor more polynucleotide sequences or two or more polypeptide sequences.The terms “percent identity” and “% identity” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm (e.g., BLAST).

An oligonucleotide that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions. Asused herein, “hybridization” or “hybridizing” refers to the process bywhich an oligonucleotide single strand anneals with a complementarystrand through base pairing under defined hybridization conditions.“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Equations forcalculating T_(m), for example, nearest-neighbor parameters, andconditions for nucleic acid hybridization are known in the art.

As used herein, “target” or “target nucleic acid” refers to a nucleicacid molecule containing a sequence that has at least partialcomplementarity with an oligonucleotide, for example, a probe or aprimer. A “target” sequence may include a part of a gene or genome.

As used herein, “nucleic acid,” “nucleotide sequence,” or “nucleic acidsequence” refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof and to naturally occurring or synthetic molecules.These terms also refer to DNA or RNA of genomic or synthetic origin,which may be single-stranded or double-stranded and may represent thesense or the antisense strand, or to any DNA-like or RNA-like material.An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose. RNA may be used in the methods described hereinand/or may be converted to cDNA by reverse transcription for use in themethods described herein.

As used herein, “amplification” or “amplifying” refers to the productionof additional copies of a nucleic acid sequence. Amplification isgenerally carried out using polymerase chain reaction (PCR) technologiesknown in the art. The term “amplification reaction system” refers to anyin vitro means for multiplying the copies of a target sequence ofnucleic acid. The term “amplification reaction mixture” refers to anaqueous solution comprising the various reagents used to amplify atarget nucleic acid. These may include enzymes (e.g., a thermostablepolymerase), aqueous buffers, salts, amplification primers, targetnucleic acid, nucleoside triphosphates, and optionally, at least onelabeled probe and/or optionally, at least one agent for determining themelting temperature of an amplified target nucleic acid (e.g., afluorescent intercalating agent that exhibits a change in fluorescencein the presence of double-stranded nucleic acid).

The amplification methods described herein may include “real-timemonitoring” or “continuous monitoring.” These terms refer to monitoringmultiple times during a cycle of PCR, preferably during temperaturetransitions, and more preferably obtaining at least one data point ineach temperature transition. The term “homogeneous detection assay” isused to describe an assay that includes coupled amplification anddetection, which may include “real-time monitoring” or “continuousmonitoring.”

Amplification of nucleic acids may include amplification of nucleicacids or subregions of these nucleic acids. For example, amplificationmay include amplifying portions of nucleic acids between 30 and 50,between 50 and 100, or between 100 and 300 bases long by selecting theproper primer sequences and using PCR. In further aspects, amplificationcan be achieved using an isothermal amplification technique (i.e.,without the need for thermal cycling). For example, methods forisothermal nucleic acid amplification, such as loop mediated isothermalamplification (LAMP), are provided in U.S. Pat. No. 6,410,278, and US.Patent Publn. 20080182312, each of which is incorporated herein byreference in its entirety.

The disclosed methods may include amplifying at least one or morenucleic acids in the sample. In the disclosed methods, amplification maybe monitored using real-time methods.

Amplification mixtures may include natural nucleotides (including A, C,G, T, and U) and non-natural or non-standard nucleotides (e.g.,including iC and iG). DNA and RNA oligonucleotides include deoxyribosesor riboses, respectively, coupled by phosphodiester bonds. Eachdeoxyribose or ribose includes a base coupled to a sugar. The basesincorporated in naturally-occurring DNA and RNA are adenosine (A),guanosine (G), thymidine (T), cytosine (C), and uridine (U). These fivebases are “natural bases.” According to the rules of base pairingelaborated by Watson and Crick, the natural bases hybridize to formpurine-pyrimidine base pairs, where G pairs with C and A pairs with T orU. These pairing rules facilitate specific hybridization of anoligonucleotide with a complementary oligonucleotide.

The formation of base pairs by natural bases is facilitated by thegeneration of two or three hydrogen bonds between the two bases of eachbase pair. Each of the bases includes two or three hydrogen bonddonor(s) and hydrogen bond acceptor(s). The hydrogen bonds of the basepair are each formed by the interaction of at least one hydrogen bonddonor on one base with a hydrogen bond acceptor on the other base.Hydrogen bond donors include, for example, heteroatoms (e.g., oxygen ornitrogen) that have at least one attached hydrogen. Hydrogen bondacceptors include, for example, heteroatoms (e.g., oxygen or nitrogen)that have a lone pair of electrons.

The natural or non-natural nucleotides used herein can be derivatized bysubstitution at non-hydrogen bonding sites to form modified natural ornon-natural nucleotides. For example, a natural nucleotide can bederivatized for attachment to a support by coupling a reactivefunctional group (for example, thiol, hydrazine, alcohol, amine, and thelike) to a non-hydrogen bonding atom of the nucleotide. Other possiblesubstituents include, for example, biotin, digoxigenin, fluorescentgroups, alkyl groups (e.g., methyl or ethyl), and the like.

The use of non-natural nucleotides according to the methods disclosedherein is extendable beyond the detection and quantification of nucleicacid sequences present in a sample. For example, non-natural nucleotidescan be recognized by many enzymes that catalyze reactions associatedwith nucleic acids. While a polymerase requires a complementarynucleotide to continue polymerizing and extending an oligonucleotidechain, other enzymes do not require a complementary nucleotide. If anon-natural nucleotide is present in the template and its complementarynon-natural nucleotide is not present in the reaction mix, a polymerasewill typically stall (or, in some instances, misincorporate a base whengiven a sufficient amount of time) when attempting to extend anelongating primer past the non-natural nucleotide. However, otherenzymes that catalyze reactions associated with nucleic acids, such asligases, kinases, nucleases, polymerases, topoisomerases, helicases, andthe like can catalyze reactions involving non-natural nucleotides. Suchfeatures of non-natural nucleotides can be taken advantage of, and arewithin the scope of the presently disclosed methods and kits.

The nucleotides disclosed herein, which may include non-naturalnucleotides, may be coupled to a label (e.g., a quencher or afluorophore). Coupling may be performed using methods known in the art.

The oligonucleotides of the present methods may function as primers. Insome embodiments, the oligonucleotides are labeled. For example, theoligonucleotides may be labeled with a reporter that emits a detectablesignal (e.g., a fluorophore). The oligonucleotides may include at leastone non-natural nucleotide. For example, the oligonucleotides mayinclude at least one nucleotide having a base that is not A, C, G, T, orU (e.g., iC or iG). Where the oligonucleotide is used as a primer forPCR, the amplification mixture may include at least one nucleotide thatis labeled with a quencher (e.g., Dabcyl). The labeled nucleotide mayinclude at least one non-natural or non-standard nucleotide. Forexample, the labeled nucleotide may include at least one nucleotidehaving a base that is not A, C, G, T, or U (e.g., iC or iG).

In some embodiments, the oligonucleotide may be designed not to form anintramolecular structure, such as a hairpin. In other embodiments, theoligonucleotide may be designed to form an intramolecular structure,such as a hairpin. For example, the oligonucleotide may be designed toform a hairpin structure that is altered after the oligonucleotidehybridizes to a target nucleic acid, and optionally, after the targetnucleic acid is amplified using the oligonucleotide as a primer.

The oligonucleotide may be labeled with a fluorophore that exhibitsquenching when incorporated in an amplified product as a primer. Inother embodiments, the oligonucleotide may emit a detectable signalafter the oligonucleotide is incorporated in an amplified product as aprimer (e.g., inherently, or by fluorescence induction or fluorescencedequenching). Such primers are known in the art (e.g., LightCyclerprimers, Amplifluor™ primers, Scorpion™ primers, and Lux™ primers). Thefluorophore used to label the oligonucleotide may emit a signal whenintercalated in double-stranded nucleic acid. As such, the fluorophoremay emit a signal after the oligonucleotide is used as a primer foramplifying the nucleic acid.

The oligonucleotides that are used in the disclosed methods may besuitable as primers for amplifying at least one nucleic acid in thesample and as probes for detecting at least one nucleic acid in thesample. In some embodiments, the oligonucleotides are labeled with atleast one fluorescent dye, which may produce a detectable signal. Thefluorescent dye may function as a fluorescence donor for fluorescenceresonance energy transfer (FRET). The detectable signal may be quenchedwhen the oligonucleotide is used to amplify a target nucleic acid. Forexample, the amplification mixture may include nucleotides that arelabeled with a quencher for the detectable signal emitted by thefluorophore. Optionally, the oligonucleotides may be labeled with asecond fluorescent dye or a quencher dye that may function as afluorescence acceptor (e.g., for FRET). Where the oligonucleotide islabeled with a first fluorescent dye and a second fluorescent dye, asignal may be detected from the first fluorescent dye, the secondfluorescent dye, or both. Signals may be detected at a gradient oftemperatures (e.g., in order to determine a melting temperature for anamplicon, a complex that includes a probe hybridized to a target nucleicacid, a hairpin, or a T probe complex).

The disclosed methods may be performed with any suitable number ofoligonucleotides. Where a plurality of oligonucleotides are used (e.g.,two or more oligonucleotides), different oligonucleotide may be labeledwith different fluorescent dyes capable of producing a detectablesignal. In some embodiments, oligonucleotides are labeled with at leastone of two different fluorescent dyes. In further embodiments,oligonucleotides are labeled with at least one of three differentfluorescent dyes.

In some embodiments, each different fluorescent dye emits a signal thatcan be distinguished from a signal emitted by any other of the differentfluorescent dyes that are used to label the oligonucleotides. Forexample, the different fluorescent dyes may have wavelength emissionmaximums all of which differ from each other by at least about 5 nm(preferably by least about 10 nm). In some embodiments, each differentfluorescent dye is excited by different wavelength energies. Forexample, the different fluorescent dyes may have wavelength absorptionmaximums all of which differ from each other by at least about 5 nm(preferably by at least about 10 nm).

Where a fluorescent dye is used to determine the melting temperature ofa nucleic acid in the method, the fluorescent dye may emit a signal thatcan be distinguished from a signal emitted by any other of the differentfluorescent dyes that are used to label the oligonucleotides. Forexample, the fluorescent dye for determining the melting temperature ofa nucleic acid may have a wavelength emission maximum that differs fromthe wavelength emission maximum of any other fluorescent dye that isused for labeling an oligonucleotide by at least about 5 nm (preferablyby least about 10 nm). In some embodiments, the fluorescent dye fordetermining the melting temperature of a nucleic acid may be excited bydifferent wavelength energy than any other of the different fluorescentdyes that are used to label the oligonucleotides. For example, thefluorescent dye for determining the melting temperature of a nucleicacid may have a wavelength absorption maximum that differs from thewavelength absorption maximum of any fluorescent dye that is used forlabeling an oligonucleotide by at least about 5 nm (preferably by leastabout 10 nm).

The methods may include determining the melting temperature of at leastone nucleic acid in a sample (e.g., an amplicon or a nucleic acidcomplex that includes a probe hybridized to a target nucleic acid),which may be used to identify the nucleic acid. Determining the meltingtemperature may include exposing an amplicon or a nucleic acid complexto a temperature gradient and observing a detectable signal from afluorophore. Optionally, where the oligonucleotides of the method arelabeled with a first fluorescent dye, determining the meltingtemperature of the detected nucleic acid may include observing a signalfrom a second fluorescent dye that is different from the firstfluorescent dye. In some embodiments, the second fluorescent dye fordetermining the melting temperature of the detected nucleic acid is anintercalating agent. Suitable intercalating agents may include, but arenot limited to SYBR™ Green 1 dye, SYBR dyes, Pico Green, SYTO dyes,SYTOX dyes, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2, ethidium derivatives, acridine, acridine orange, acridinederivatives, ethidium-acridine heterodimer, ethidium monoazide,propidium iodide, cyanine monomers, 7-aminoactinomycin D, YOYO-1,TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1,cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5,PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, and mixturesthereof. In suitable embodiments, the selected intercalating agent isSYBR™ Green 1 dye.

In the disclosed methods, each of the amplified target nucleic acids orreporter probe-template pairs may have different melting temperatures.For example, each of the amplified target nucleic acids or reporterprobe-template pairs may have melting temperatures that differ by 1-10°C., for example, at least about 1° C., more preferably by at least about2° C., or even more preferably by at least about 4° C. from the meltingtemperature of any of the other amplified target nucleic acids orreporter probe-template pairs.

As used herein, “labels” or “reporter molecules” are chemical orbiochemical moieties useful for labeling a nucleic acid. “Labels” and“reporter molecules” include fluorescent agents, chemiluminescentagents, chromogenic agents, quenching agents, radionuclides, enzymes,substrates, cofactors, scintillation agents, inhibitors, magneticparticles, and other moieties known in the art. “Labels” or “reportermolecules” are capable of generating a measurable signal and may becovalently or noncovalently joined to an oligonucleotide.

As used herein, a “fluorescent dye” or a “fluorophore” is a chemicalgroup that can be excited by light to emit fluorescence. Some suitablefluorophores may be excited by light to emit phosphorescence. Dyes mayinclude acceptor dyes that are capable of quenching a fluorescent signalfrom a fluorescent donor dye. Dyes that may be used in the disclosedmethods include, but are not limited to, fluorophores such as, a redfluorescent squarine dye such as 2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dio-xolate, aninfrared dye such as 2,4Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,-3-dioxolate,or an orange fluorescent squarine dye such as2,4-Bis[3,5-dimethyl-2-pyrrolyl]cyclobutenediylium-1,3-diololate.Additional non-limiting examples of fluorophores include quantum dots,Alexa Fluor™ dyes, AMCA, BODIPY™ 630/650, BODIPY™ 650/665, BODIPY™-FL,BODIPY™-R6G, BODIPY™-TMR, BODIPY™-TRX, Cascade Blue™ CyDye™, includingbut not limited to Cy2™, Cy3™, and Cy5™, a DNA intercalating dye,6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green™ 488, Oregon Green™ 500,Oregon Green™ 514, Pacific Blue™, REG, phycobilliproteins including, butnot limited to, phycoerythrin and allophycocyanin, Rhodamine Green™,Rhodamine Red™, ROX™ TAMRA™, TET™, Tetramethylrhodamine, or Texas Red™.

Fluorescent dyes or fluorophores may include derivatives that have beenmodified to facilitate conjugation to another reactive molecule. Assuch, fluorescent dyes or fluorophores may include amine-reactivederivatives, such as isothiocyanate derivatives and/or succinimidylester derivatives of the fluorophore.

The oligonucleotides and nucleotides of the disclosed methods may belabeled with a quencher. Quenching may include dynamic quenching (e.g.,by FRET), static quenching, or both. Suitable quenchers may includeDabcyl. Suitable quenchers may also include dark quenchers, which mayinclude black hole quenchers sold under the trade name “BHQ” (e.g.,BHQ-0, BHQ-1, BHQ-2, and BHQ-3, Biosearch Technologies, Novato, Calif.).Dark quenchers also may include quenchers sold under the trade name“QXL™” (Anaspec, San Jose, Calif.). Dark quenchers also may includeDNP-type non-fluorophores that include a 2,4-dinitrophenyl group.

The methods and compositions disclosed herein may be used incompartmentalized reactions. One approach for compartmentalizingreactions is by using droplets, which are isolated volumes of a firstfluid that are completely surrounded by a second fluid or by a secondfluid and one or more surfaces. In the molecular diagnostics and lifescience research fields this is typically two immiscible liquids.Various embodiments disclosed herein employ a water-in-oil emulsioncomprising a plurality of aqueous droplets in a non-aqueous continuousphase. All or a subset of the aqueous droplets may contain an analyte ofinterest. Emulsions are formed by combining two immiscible phases (e.g.,water and oil), often in the presence of one or more surfactants. Basictypes of emulsions are oil-in-water (o/w), water-in-oil (w/o), andbi-continuous. In droplet-based biological assays, the emulsion willtypically be a water-in-oil emulsion with the assay reagents (e.g., PCRprimers, salts, enzymes, etc.) contained in the aqueous phase. The “oil”phase may be a single oil or a mixture of different oils. Any suitablenon-aqueous fluid may form the non-aqueous continuous phase of theemulsions disclosed herein. In some embodiments, the non-aqueouscontinuous phase comprises a mineral oil, a silicone oil, or afluorinated oil (e.g., Fluorinert® FC-40 [Sigma-Aldrich]).

The droplets may be imaged by a variety of techniques. To facilitateimaging, the composition containing the droplets may be dispersed on asurface such that the droplets are disposed substantially in a monolayeron the surface. The imaging surface may be, for example, on a slide orin a chamber, such as a glass or quartz chamber. The droplets, as wellas labeled analytes or reaction products (e.g., hairpin probes) withinthe droplets, may be detected using an imaging system. For example,detection may comprise imaging fluorescent wavelengths and/orfluorescent intensities emitted from the labeled hairpin probes. Inembodiments where the droplets also contain encoded particles, such asencoded microspheres, the imaging may comprise taking a decoding imageof the encoded particles and taking an assay imaging to detect theprobes in the droplets. A comparison of the decoding image and the assayimage permits greater multiplex capabilities by using combinations offluorophores. The methods of the present invention may further comprisecorrelating the signal from the directly or indirectly labeledamplification product with the concentration of DNA or RNA in a sample.Examples of imaging systems that could be adapted for use with themethods and compositions disclosed herein are described in U.S. Pat. No.8,296,088 and U.S. Pat. Publ. 2012/0288897, which are incorporatedherein by reference.

As discussed above, the polymerase chain reaction (PCR) is an example ofa reaction that may be performed within a droplet. In particular,droplets are useful in digital PCR (dPCR) techniques. dPCR involvespartitioning the sample such that individual nucleic acid moleculescontained in the sample are localized in many separate regions, such asin individual wells in microwell plates, in the dispersed phase of anemulsion, or arrays of nucleic acid binding surfaces. Each partition(e.g., droplet) will contain 0 or greater than zero molecules, providinga negative or positive reaction, respectively. Unlike conventional PCR,dPCR is not dependent on the number of amplification cycles to determinethe initial amount of the target nucleic acid in the sample.Accordingly, dPCR eliminates the reliance on exponential data toquantify target nucleic acids and provides absolute quantification. Beademulsion PCR, which clonally amplifies nucleic acids on beads in anemulsion, is one example of a dPCR technique in which the reactions areportioned into droplets. See, e.g., U.S. Pat. Nos. 8,048,627 and7,842,457, which are hereby incorporated by reference. When dPCR isperformed in an emulsion as discussed in more detail below, the emulsionshould be heat stable to allow it to withstand thermal cyclingconditions.

There are various ways of performing dPCR in an emulsion. For example,in one approach a DNA sample is diluted to an appropriate concentration,mixed with PCR reagents (primers, dNTPs, etc.) and encapsulated indroplets in an emulsion as described above, resulting in a number ofdiscrete reaction samples. The droplets are subjected to PCR thermalcycling and the amplicons detected by florescence (or other suitablereporter) imaging as described above. In the context of the presentcleavable probe embodiments, the amplicons are detected by florescence(or other suitable reporter) of the probes.

The thermal cycling of the droplets may be performed by any suitabletechnique known in the art. For example, the droplets may be thermalcycled in a tube or chamber than can be heated and cooled. In someembodiments, the methods employ continuous-flow amplification to amplifythe nucleic acid template. Various methods of continuous flowamplification have been reported. For example, U.S. Pat. No. 7,927,797,which in incorporated herein by reference, describes a water-in-oilemulsion used in conjunction with a continuous flow PCR. Isothermalreactions (e.g., rolling circle amplification, whole genomeamplification, NASBA, or strand displacement amplification) may also beperformed in droplets. The system may also be used to monitor thedroplets while increasing or decreasing the temperature to obtain meltprofiles per droplet, which will allow for multiplexed detection andquantification. The probes themselves may be used within droplets toisothermally amplify signal such that other forms of amplification suchas PCR or other isothermal amplification reactions are not necessary todetect low copy numbers of target within a droplet.

II. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Multiprobe Probe Systems

Solution phase multiplexing strategies for molecular assays rely on theuse of multiple fluorophores in conjunction with generation of multiplefluorescence melt curves for detection of >10 targets. Variousembodiments disclosed herein provide a real time probe based chemistrythat allows higher multiplexing capabilities to be achieved by utilizingextendable hairpin probes to create multiple melt curves per channel. Anexample of probes for use in this system is shown in FIG. 1A. In thisexample, the cleavable probe comprises a reporter-labeled isoGnucleotide (“isoG*”) at its 5′ end, a first sequence region (“Tag A”), asecond sequence region (“Tag B”), a loop sequence, a sequence regionthat is the reverse compliment of Tag B (“Tag B complement”); and asequence complementary to the target amplicon (indicated as “A”). Thecleavable probe also comprises one or more ribonucleotides (indicated bythe solid square) in the “A” sequence and may comprise a modificationthat blocks extension on the 3′ end (indicated as “P”). In the presenceof a target amplicon, the cleavable probe hybridizes to the amplicon andis cleaved at the ribonucleotide position by RNase H2 (which recognizesand cleaves ribonucleotides in an annealed RNA/DNA hybrid). Followingcleavage, the probe can hybridize to itself via the Tag B and Tag Bcomplement sequences to form a hairpin. Extension of the probe willsynthesize sequences complementary to the Tag A sequences and willincorporate a quencher labeled isoC (“isoC^(Q)”). The resulting hairpinprobe quenches the fluorescence of the labeled isoG. The probes can bedesigned to have unique melt temperatures (T_(m)), such as by adjustingthe sequence and length of the first and second sequence regions. Thus,the composition and length of the Tag A and Tag B stem structures can bevaried to resolve in any desired melt temperature for the hairpin probe(see, FIG. 1B).

Materials and Methods Probe Design Parameters

Multiple constructs of cleavable probes were designed without a targetsequence specific tail (post cleavage) to determine optimal designparameters for an extendable hairpin. The targeted T_(m) for thesequence specific tail was 10° C. above the reaction temperature (˜58°C.). The hairpin constructs were designed to have a T_(m)>60° C. toallow for the formation of the unimolecular structure post cleavage ofRNA/DNA hybrid. These constructs were designed to determine requirementsfor loop size (number of bases), stem size, Gibbs free energy and T_(m)of the hairpin post cleavage. Examples of specific probes that wereconstructed are shown (FIG. 2). For these proof of concept experiments,a loop of multi-adenine residues ending with two cytosine “clamps” ateach side of the loop was used (sequence between the font in italics).

1. Folding of the Probes

Temperature gradient was used to assess the folding profile of theseconstructs by monitoring the decrease of fluorescence intensity of thehairpin over temperatures ranging from 95° C. to 41° C. The constructsof FIG. 2 were added to a reaction mixture containing BTP-KCl pH 9.1buffer, 2.5 mM dNTPs, 2.5 mM MgCl₂, 1 mM Dabcyl-isoG and Titanium Taqenzyme (Clontech). After initial denaturation step at 95° C., thereaction temperature was decreased from 95° C. to 41° C. by 3° C.increments with a hold of 10 seconds at each interval. The temperatureat which complete quenching was observed for each construct was recordedas the folding temperature of the hairpin.

2. Efficiency of Hairpin-Loop Formation: Folding, Extending andQuenching of the Probes

The efficiency of the hairpin formation was evaluated by measuring rateof quenching of each construct at 3 temperatures. Constructs of FIG. 2were added to a reaction mixture containing BTP-KC1 pH 9.1 buffer, 2.5mM dNTPs, 1mM isoG-dabcyl- and Titanium Taq enzyme (Clontech). After 2minutes of activation step at 95° C., the reaction was incubated at 50°C., 62° C. and 68° C. for 30 minutes to allow for hairpins to fold,extend and incorporate isoG-dabcyl . This was followed by a melt curvecycling protocol of 60° C. 30 s and incremental increase to 95° C. Theefficiency of the reaction was determined by the Ct values generatedwhen quenching was achieved.

3. Single Plex RT-PCR with Full Length Probes

Feasibility of using the multiprobe RTx probes for detection in anamplification reaction was first evaluated in a singleplex RT-PCRreaction. Multiple designs of the full length probes (with sequencespecific tail) were generated based on the hairpin designs assessed in(FIGS. 2-3). The target T_(m) for the sequence specific segment was −10°C. higher than the annealing temperature of the reaction. The sequenceof the primers (Table 1) and probes were based on the matrix gene ofInfluenza B virus. Nucleic acid extracted from Influenza B Strain:B/Malaysia/2506/04 (Zeptometrix) was used as a template in a one-stepRT-PCR reaction. Specifically, PCR primers (forward 180 nM, reverse 60nM) and probe (120 nM) were added to a reaction mixture containingBTP-KCl pH 9.1 buffer, 2.5 mM dNTPs, 2.5 mM MgCl₂, 1 mM Dabcyl-isoG,Titanium Taq enzyme (Clontech) and MMLV (Promega) and RNase H (IDT). Thefollowing cycling conditions were used for amplification and melt curveanalysis: 50° C. , 5 minutes; 95° C. for 10 minutes; 95° C. for 10 s,58° C. for 20 s for 45 cycles followed by a melt program of 60° C. for30 s and 95° C. is ending with a cooling step at 40° C.

TABLE 1 PCR primers. T_(m) Primer name Sequence (° C.) FluB Fwd-shortGAA GCA TTT GAA ATA 61   GCA GAA GG (SEQ ID NO: 22) FluB Rev-shortCAC AGA GCG TTC CTA 62.8 GTT TTA CT (SEQ ID NO: 23)

Results Melt Profile of Hairpin Loops

The melt profile of the hairpin probes of FIG. 2 were generated todetermine the folding temperature of various constructs. This wasmeasured by monitoring drop in fluorescence intensity over a temperaturegradient of 95° C. to 41° C. (FIG. 4). Graphs showing the quenchingprofile for three exemplary constructs RTx-5, RTx-10 and RTx-11 areshown in FIGS. 5-7, respectively. The results of all studies are shownin Table 2, below. It was found that hairpin constructs RTx-1, 2, 3, 5,6, 7, and 8 are completely quenched by 71° C. temperature stepcorresponding to calculated T_(m) of the extended hairpin ˜71° C. (IDT).Hairpin constructs RTx-4, 9 and 10 are quenched by the 62° C. andhairpin RTx-11 at 41° C.

TABLE 2 Summary of folding temperature for various hairpin probes.deltaG Tm stem loop Construct Folding Temperature (° C.) Stem (bp) loop(bp) (kcal · mole−1) (° C.) % GC RTx-1 71 8 7 −1.27 64.8 50 RTx-2 8 12−1.06 63.6 50 RTx-3 7 7 −0.87 63.3 57 RTx-5 9 7 −1.62 65.7 44 RTx-6 9 12−1.41 64.6 44 RTx-7 8 7 −0.93 62.9 50 RTx-8 8 12 −0.72 61.8 50 RTx-4 627 12 −0.66 62 57 RTx-9 6 7 −0.26 59.8 67 RTx-10 6 7 −0.53 61.7 67 RTx-1141 5 7 −0.5 62 80

When the T_(m) of stem loop, deltaG values, loop size and stem size arecompared the data suggests that the main factor influencing theformation of the hairpin is the number of bases in the stem. Thesecondary factor may be the Gibbs Free energy associated with thefolding of the hairpin as the delta G of the constructs with the correctfolding T_(m) are lower than the constructs with T_(m)'s of 62° C. and41° C.

Efficiency of Hairpin Loop Formation Amplification

The hairpin constructs from FIG. 2 were used to determine the efficiencyof hairpin formation at various temperatures. The results confirm theobservations made in above. The reaction rates are very fast for probesRT-x 1, 2, 7, and 8 (FIG. 8 and FIG. 9). Slower reaction rates wereobserved for probes RTx-9 and 10, Ct values ranged from 5-10 and thehighest Ct value recorded for complete quenching was for RTx 11, 30-35(FIG. 10). These hairpins require lower temperatures to form and extend,which translates into longer times required for folding.

Melt Curve Analysis

Melt curves analysis of hairpin constructs RTx 1, 2, 3, and 4 show thatincreasing temperature results in sharper melt curves (FIG. 8). This isaccompanied by slight shift in the recorded T_(m). Sharper melt curveswere likewise generated at 62° C. with constructs RT-x 5 and 6, and noshift in T_(m) is observed. Melt curves and T_(m)s of constructs RTx-7and 8 deviate from the trend observed for the other hairpins (FIG. 9).Wide and overlapping melt curves were generated with constructs RTx 9,10, and 11, (FIG. 10) corresponding to data generated above.

Single Plex RT-PCR with Full Length Probes

Full length probe designs were created based on the data generated onthe hairpin constructs from FIG. 2 and FIG. 3. Minimum stem sizetargeted was 8 bases, 12-20 residues for the loop and a T_(m) of 55°C.-66.4° C. for the hairpin loop.

Detection

All probes generated Ct values in the range of 34-35 Cts (FIGS.11A-11D). Melt curves of most probes indicated the presence of onespecies, mainly the extended hairpin. Minor, high T_(m) peaks weredetected for some of the probes.

The same fluorescence intensity was recorded for all the probes with theexception of FL-RTx-2-12AT1 and FL-RTx-2-12AT2. The calculated hairpinloop T_(m) of these probes is very close to the reaction temperature(58° C.). Reducing the reaction temperature may improve the number ofhairpin molecules formed and provide better detection.

Specificity

Two negative controls were included (FIGS. 11A-11D). The purpose forincluding a template negative control (water) was to detect formation ofthe hairpin non-specifically due to cleavage of the full length probe byRNase H2. Only probe FL-RTx-2-12-AT-4 (FIG. 11D) showed backgroundnon-specific melt curve, the same size as the hairpin which mightsuggest non-specific cleavage of the probe. The second negative controlused was clinical negative specimen collected from asymptomaticpatients. The objective was to evaluate the specificity of the probe inthe presence of unrelated template. None of the probes showed anynon-specific interaction with the template.

Example 2—Additional Hairpin Probe Detection Systems

A further example of a hairpin probe detection system is shown in FIG.12. The reporter probe comprises a reporter-labeled isoC nucleotide(“isoC*”) at its 5′ end, a first sequence region (“region 1”), asequence that includes isoG and/or isoC positions (the “isoprimer”); anda sequence complementary to the amplicon (indicated as “A”). Thesequence that is complementary to the amplicon also includes at leastone ribonucleotide position. In the presence of a target amplicon thereporter probe hybridizes to the amplicon and is cleaved at theribonucleotide position by RNase H. Following cleavage, the reporterprobe can hybridize to a capture oligonucleotide (“capture oligo”),which comprises a capture segment complimentary to the isoprimer and,optionally, a portion that “A” sequence, followed by a mirror region 1and a 3′ unlabeled isoC. Extension of the reporter probe will synthesizesequences complementary to the mirror tag on the capture oligo and willincorporate a quencher labeled isoG (“isoG^(Q)”). The extended reporterprobe now includes a tag and tag complement sequence, which allows theprobe to form a hairpin and thereby quench the fluorescence of thelabeled isoC. The probes can be designed to have unique melttemperatures (T_(m)), such as by adjusting the sequence and length ofthe first sequence region. Thus, a melt analysis can be performed todifferentiation probes having different melt temperatures (and thusunquenching at different temperatures).

The assay system of FIG. 12 may also be further modified, such that thecapture probe does not require the isobase. In this system, the reporterprobe comprises a reporter-labeled isoC nucleotide (“isoC*”) at its 5′end, a first sequence region (“region 1”), a sequence that includes isoGand/or isoC positions (the “isoprimer”); and a sequence complementary tothe amplicon (indicated as “A”). The sequence that is complementary tothe amplicon also includes at least one ribonucleotide position. In thepresence of a target amplicon the reporter probe hybridizes to theamplicon and is cleaved at the ribonucleotide position by RNase H.Following cleavage, the reporter probe can hybridize to a captureoligonucleotide (“capture oligo”), which comprises a capture segmentcomplimentary to the isoprimer and, optionally, a portion that “A”sequence, followed by a mirror region 1 (which is identical to part ofthe of the region 1 sequence). Extension of the reporter probe willsynthesize sequences complementary to the mirror region 1 on the captureoligo. The cleavable probe can then form a hairpin by base pairing ofthe region 1 sequence with the sequence that is complementary to themirror region 1. Further extension of the hairpin sequence and willincorporate a quencher labeled isoG (“isoG^(Q)”). The probes can bedesigned to have unique melt temperatures (T_(m)), such as by adjustingthe sequence and length of the first sequence region. Thus, a meltanalysis can be performed to differentiation probes having differentmelt temperatures (and thus unquenching at different temperatures).

FIG. 15 shows another embodiment in which the probe comprises both afluorophore (F) and a quencher (Q). The conformation of the firstsequence region when single-stranded is such that the proximity of thefluorophore to the quencher results in detectable quenching of thesignal from the fluorophore. In the presence of a target, the probehybridizes to the target and is cleaved at the ribonucleotide positionby a ribonuclease. In this particular embodiment, the second sequenceregion complement, the ribonucleotide(s), and the target specific region3′ of the ribonucleotide(s) are complementary to the target. Followingcleavage of the probe, the second sequence region and the secondsequence region complement of the cleaved probe hybridize to each otherto form a hairpin structure. Extension of the 3′ end of the cleavedprobe onto the first sequence region creates a double-stranded moleculehaving a conformation that places the fluorophore at a greater distancefrom the quencher such that a detectable change in the signal can beobserved.

FIG. 16 shows another embodiment in which the probe comprises both afluorophore (F) and a quencher (Q). The conformation of the firstsequence region when single-stranded is such that the proximity of thefluorophore to the quencher results in detectable quenching of thesignal from the fluorophore. In the presence of a target, the probehybridizes to the target and is cleaved by the 5′ nuclease activity of apolymerase extending an upstream primer. In this particular embodiment,the second sequence region complement is not complementary to thetarget. Following cleavage of the probe, the second sequence region andthe second sequence region complement of the cleaved probe hybridize toeach other to form a hairpin structure. Extension of the 3′ end of thecleaved probe onto the first sequence region creates a double-strandedmolecule having a conformation that places the fluorophore at a greaterdistance from the quencher such that a detectable change in the signalcan be observed.

FIG. 17 shows an embodiment in which the probe comprises one member of areporter-quencher pair, in this particular case it is a fluorophore (F).In addition, the probe comprises a first sequence region, a secondsequence region, a loop region, a second sequence region complement, oneor more ribonucleotide(s), and a target specific region 3′ of theribonucleotide(s). In this particular embodiment, the loop region, thesecond sequence region complement, the ribonucleotide(s), and the targetspecific region 3′ of the ribonucleotide(s) are complementary to thetarget.

Example 3—Use of Hairpin Probes with Extension Blockers in ReverseTranscription PCR

Fwd and Rev primers were combined in a well with either ATG0015 probe orT-FL-RTx2c probe, which differed only in that ATG00015 probe contained a3 Carbon spacer (iSpC3) in the loop region and T-FL-RTx2c did not. Thesewere combined with PCR master mix and thermal cycled followed by a meltanalysis.

ATG0015:  (SEQ ID NO: 24)/56-FAM//iMe-isodC/ATATCAGTCATTTGCCCAAAA/iSpC3/ (SEQ ID NO: 25)AAACCGCAAATGAC rCAT GAG ACA GTA TAG TAG CGC TGA/ 3SpC3/ T-FL-RTx2c: (SEQ ID NO: 15) /56-FAM//iMe-isodC/ATATCAGTCATTTGCCCAAAAAAAACCGCAAATGAC rCAT GAG ACA GTA TAG TAG CGC TGA/ 3SpC3/ Fwd Primer-  (SEQ ID NO: 22) GAA GCA TTT GAA ATA GCA GAA GG Rev Primer-  (SEQ ID NO: 23) CAC AGA GCG TTC CTA GTT TTA CT

Reverse transcription PCR was performed without template to monitor fornon-specific interactions that would cause a change in signal during amelt analysis. The below PCR master mix was created for a 25 μL reactionand run on an ABI Fast 7500 real-time thermal cycler. The thermalprofile included 50° C. hold for 5 m., 95° C. hold for 2 m. 20 s., 44cycles of 95° C. for 10 s. and 57° C. for 23 s. The melt analysisincluded ramping from 60 to 95° C. and reading at every 0.5° C.

TABLE 3 PCR Master Mix Working Reagent Concentration Nuclease Free Water10X ISOlution 1× 100 mM MgCl2 2.5 mM 1M KCl 0.05M FluB Fwd primer 0.12MFluB Rev primer 0.06M Probe 0.06M RNase H2 HotStart 1 mU Glycerol FreeTitanium 1× Taq MMLV Reverse 0.75 Transcriptase

FIG. 13 shows the inverted derivative of the data obtained during themelt analysis. A non-specific melt peak at 77° C. is present for theT-FL-RTx2c probe, which lacks the 3-carbon spacer in the loop region.

Without wishing to be bound by theory, it is thought that during the lowtemperature reverse transcriptase step at 50° C., the Rev primer in thiscase is hybridizing to the probe downstream of the loop region, whichallows the primer to extend through the loop and incorporate a quencheracross from the labeled isobase. This hybridization also causes theribobase to be cleaved, allowing the probe to also extend along theprimer. The double stranded product is amplified during the PCRreaction. The extension blocker prevents the non-specific extension ofthe primer across the loop region, which not only prevents the formationof a quencher/fluorophore pair, but also prevents a double strandedproduct with sufficient Tm to be amplified during the 58° C. annealingsteps in PCR.

Example 4—Multiplexing Using a Single Dye

This study demonstrated the ability to use multiple hairpin probeshaving the same fluorophore, but differing in the Tm of the variousextended hairpin probes. Three different probes specific for eitherInfluenza A, Influenza B, or Adenovirus; having the same fluorophore(FAM), were tested together in the same PCR tube. Positive controlsamples containing extracted viral cultures of either Influenza A,Influenza B, or Adenovirus were placed in individual PCR tubescontaining the multiplex PCR reaction components. These targets weretested at 1000 copies per reaction. The cleavable probe sequences areshown in Table 4.

TABLE 4 Probe Sequences Target Name Cleavage Probe Sequence (5′-to 3′)FluB /56-FAM//iMe-isodC/CAA AAA AAA  GTCATGTTA CCAAAA(SEQ ID NO: 26)/iSpC3/AAACC TA ACATGACrCATGAGACA GTATAGTAGCG(SEQ ID NO: 27)/ 3SpC3/ FluA/56-FAM//iMe-isodC/C ATA TCA TCA  TCA TCT C ATTTTAGGC CCAAAA(SEQ ID NO: 28)/iSpC3/AAACC GCCTAAAATrCCCCTTAGTCAGAGGTGAC(SEQ ID NO: 29)/ 3SpC3/ Adeno/56-FAM//iMe-isodC/C TCC ATC CTC CTC   CTC CTC TCT CTTCGAGA CCAAAA(SEQ ID NO: 30)/iSpC3/AAACC TCT CGAAG rCGTCCTGTCCGGC(SEQ ID NO: 31)/3SpC3/

The below PCR master mix (Table 5) was created for a 25 μL reaction andrun on an Life Technologies Quant Studio real-time PCR thermal cycler.The thermal profile included 50° C. hold for 5 m., 95° C. hold for 2 m.20 s., 44 cycles of 95° C. for 10 s. and 57° C. for 23 s. The meltanalysis included ramping from 60 to 95° C. and reading at every 0.5° C.

TABLE 5 PCR Master Mix Final Reagent Concentration Nuclease Free Water10X ISOlution 1× 1M KCl 0.05M MgCl2 2.5 mM Tris pH 8.0 10 mMBisTrisPropane 10 mM Fwd primers 0.48M Rev primers 0.12M Probes 0.02MRNase H2 HotStart (I.D.T) 4 mU/μL 50× Glycerol Free Titanium 1× Taq(Clonetech) MMLV Reverse Transcriptase 2 U/μl (Promega)

FIG. 14 shows melt profile data for 6 individual reactions (1 positivefor each of the three targets at 1000 copies/reaction, and 3 No TemplateControl (NTC) samples) using the same multiplex PCR reaction mix. As canbe seen in FIG. 14, each of the FluA, FluB, and Adeno-specific cleavableprobes generated distinct melt profiles in the same fluorescencechannel. Accordingly, in this example three different viruses weredistinguished by melt profile when using the same fluorescent label.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Pat. Nos. 4,942,124; 4,284,412; 4,989,977; 4,498,766; 5,478,722;4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; 4,661,913;5,654,413; 5,656,493; 5,716,784; 5,736,330; 5,837,832; 5,837,860;5,981,180; 5,994,056; 5,736,330; 5,981,180; 6,057,107; 6,030,787;6,046,807; 6,057,107; 6,103,463; 6,139,800; 6,174,670; 6,268,222;6,322,971; 6,366,354; 6,410,278; 6,411,904; 6,449,562; 6,514,295;6,524,793; 6,528,165; 6,592,822; 6,939,720; 6,977,161; 7,226,737;7,645,868; and 7,955,802

U.S. Published Publication Nos. 2005/0191625; 2008/0182312; and2009/0148849

McMinn et al., J. Am. Chem. Soc., 121:11585, 1999.

Ren et al., J. Am. Chem. Soc., 118:1671, 1996.

Vogelstein et al., P.C.R. Digital, Proc. Natl. Acad. Sci. USA,96:9236-9241, 1996.

Yan et al., “Isothermal Amplified Detection of DNA and RNA” Mol.GioSyst. 10:970-1003, 2014.

What is claimed is:
 1. A method for detecting the presence of a targetnucleic acid comprising: (a) contacting the sample with a firstcleavable probe, said probe comprising, from 5′ to 3′, (i) a firstsequence region comprising at least one non-natural nucleotide labeledwith a first member of a reporter-quencher pair; (ii) a second sequenceregion; (iii) a sequence that is the reverse complement of the secondsequence region; and (iv) a sequence that is complementary to a firstregion on a first strand of the target nucleic acid; (b) hybridizing thecleavable probe and an upstream primer to the target nucleic acid, andperforming extension using a polymerase possessing 5′ nuclease activity;(c) extending the upstream primer until contacting the cleavable probewith the polymerase possessing nuclease activity, thereby cleaving theprobe that is hybridized to the target nucleic acid to form a truncatedcleaved probe; (d) allowing the truncated cleaved probe to hybridize toitself to form a hairpin probe; (e) extending the hairpin probe in thepresence of a non-natural nucleotide that is labeled with a secondmember of the reporter-quencher pair and is capable of base-pairing withthe at least one non-natural nucleotide of the first sequence region;and (f) detecting the target nucleic acid by detecting a change insignal from the label on the extended hairpin probe.
 2. The method ofclaim 1, further comprising performing melt analysis on the extendedhairpin probe.
 3. The method of claim 1 further including a loopsequence of one or more nucleotides between the second sequence regionand the sequence that is the reverse complement of the second sequenceregion.
 4. The method of claim 1 wherein the cleavable probe comprisesan extension blocking modification at the 3′ end.
 5. The method of claim1 wherein detecting the presence of the target nucleic acid comprisesdetecting a change in signal from the reporter as the temperature isincreased above the melt point of the extended hairpin probe.
 6. Amethod for detecting the presence of a target nucleic acid comprising:(a) contacting the sample with a first cleavable probe, said probecomprising, from 5′ to 3′, (i) a first sequence region labeled with areporter-quencher pair; (ii) a second sequence region; (iii) a sequencethat is the reverse complement of the second sequence region; and (iv) asequence that is complementary to a first region on a first strand ofthe target nucleic acid; (b) hybridizing the cleavable probe and anupstream primer to the target nucleic acid, and performing extensionusing a polymerase possessing 5′ nuclease activity; (c) extending theupstream primer until contacting the cleavable probe with the polymerasepossessing nuclease activity, thereby cleaving the probe that ishybridized to the target nucleic acid to form a truncated cleaved probe;(d) allowing the truncated cleaved probe to hybridize to itself to forma hairpin probe; (e) extending the hairpin probe using the firstsequence region as a template to cause the flurophore and quencher to bephysically separated; and (f) detecting the target nucleic acid bydetecting a change in signal from the extended hairpin probe.
 7. Themethod of claim 6 further including a loop sequence of one or morenucleotides between the second sequence region and the sequence that isthe reverse complement of the second sequence region.
 8. The method ofclaim 6 wherein the cleavable probe comprises an extension blockingmodification at the 3′ end.
 9. The method of claim 6 wherein detectingthe presence of the target nucleic acid comprises detecting a change insignal from the reporter as the temperature is increased above the meltpoint of the extended hairpin probe.
 10. The method of claim 6 furthercomprising performing melt analysis on the extended hairpin probe.